Methods and compositions for making antibodies and antibody derivatives with reduced core fucosylation

ABSTRACT

The invention provides methods and compositions for preparing antibodies and antibody derivatives with reduced core fucosylation.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/632,925 filed Feb. 26, 2015, which is a continuation of U.S. patentapplication Ser. No. 14/043,742 filed Oct. 1, 2013 (now U.S. Pat. No.8,993,326), which is a divisional of Ser. No. 13/405,143 filed Feb. 24,2012 (now U.S. Pat. No. 8,574,907), which is a divisional of Ser. No.12/434,533 filed May 1, 2009 (now U.S. Pat. No. 8,163,551), which claimsthe benefit of U.S. Provisional Application No. 61/050,173 filed May 2,2008 and U.S. Provisional Application No. 61/092,700 filed Aug. 28, 2008and U.S. Provisional Application No. 61/107,289 filed Oct. 21, 2008, thecontents of each are incorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

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REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

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BACKGROUND OF THE INVENTION

Recombinant therapeutic proteins are produced by many different methods.One preferred method is production of recombinant proteins frommammalian host cell lines. Cell lines, such as Chinese Hamster Ovary(CHO) cells, are engineered to express the therapeutic protein ofinterest. Different cell lines have advantages and disadvantages forrecombinant protein production, including protein characteristics andproductivity. Selection of a cell line for commercial production oftenbalances the need for high productivity with the ability to deliverconsistent product quality with the attributes required of a givenproduct. One important class of therapeutic recombinant proteins thatrequire consistent, high quality characteristics and high titerprocesses are monoclonal antibodies.

Monoclonal antibodies produced in mammalian host cells can have avariety of post-translational modifications, including glycosylation.Monoclonal antibodies, such as IgG1s, have an N-linked glycosylationsite at asparagine 297 (Asn297) of each heavy chain (two per intactantibody). The glycans attached to Asn297 on antibodies are typicallycomplex biantennary structures with very low or no bisectingN-acetylglucosamine (bisecting GIcNAc) with low amounts of terminalsialic acid and variable amounts of galactose. The glycans also usuallyhave high levels of core fucosylation. Reduction of core fucosylation inantibodies has been shown to alter Fc effector functions, in particularFcgamma receptor binding and ADCC activity. This observation has lead tointerest in the engineering cell lines so they produce antibodies withreduced core fucosylation.

Methods for engineering cell lines to reduce core fucosylation includedgene knock-outs, gene knock-ins and RNA interference (RNAi). In geneknock-outs, the gene encoding FUT8 (alpha 1,6-fucosyltransferase enzyme)is inactivated. FUT8 catalyzes the transfer of a fucosyl residue fromGDP-fucose to position 6 of Asn-linked (N-linked) GlcNac of an N-glycan.FUT8 is reported to be the only enzyme responsible for adding fucose tothe N-linked biantennary carbohydrate at Asn297. Gene knock-ins addgenes encoding enzymes such as GNTIII or a golgi alpha mannosidase II.An increase in the levels of such enzymes in cells diverts monoclonalantibodies from the fucosylation pathway (leading to decreased corefucosylation), and having increased amount of bisectingN-acetylglucosamines. RNAi typically also targets FUT8 gene expression,leading to decreased mRNA transcript levels or knock out gene expressionentirely.

Alternatives to engineering cell lines include the use of small moleculeinhibitors that act on enzymes in the glycosylation pathway. Inhibitorssuch as catanospermine act early in the glycosylation pathway, producingantibodies with immature glycans (e.g., high levels of mannose) and lowfucosylation levels. Antibodies produced by such methods generally lackthe complex N-linked glycan structure associated with mature antibodies.

In contrast, the present invention provides small molecule fucoseanalogs for use in producing recombinant antibodies that have complexN-linked glycans, but have reduced core fucosylation.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for preparing antibodiesand antibody derivatives with reduced core fucosylation. The methods andcompositions are premised in part on the unexpected results presented inthe Examples showing that culturing host cells, expressing an antibodyor antibody derivative, in the presence of a fucose analog (havingformula I, II, III, IV, V or VI) produces an antibody having reducedcore fucosylation (i.e., reduced fucosylation of N-acetylglucosamine ofthe complex N-glycoside-linked sugar chains bound to the Fc regionthrough the N-acetylglucosamine of the reducing terminal of the sugarchains). Such antibodies and antibody derivatives may exhibit increasedeffector function (ADCC), as compared with antibodies or antibodyderivatives produced from such host cells cultured in the absence of thefucose analog.

In another aspect, compositions of antibodies and antibody derivativesare provided. The antibodies and antibody derivatives can be produced bythe methods described herein.

In another aspect, fucose analogs are provided. The fucose analogs canbe added to mammalian cell culture media to inhibit or reduce corefucosylation. Also provided is cell culture media comprising aneffective amount of such a fucose analog(s).

These and other aspects of the present invention may be more fullyunderstood by reference to the following detailed description,non-limiting examples of specific embodiments, and the appended figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an analysis (electropherograms) of glycans isolated from ananti-CD70 antibody (h1F6) produced from control (A) and alkynyl fucoseperacetate (AlkF)-treated host cells (B and C). The panels show theidentity and relative distribution of glycans. “G0” refers to thecarbohydrate structure where there is no galactose at the twonon-reducing termini. “G1” refers to a carbohydrate structure where oneof the non-reducing termini has a galactose (a mixture of two isomers).“G2” refers to a carbohydrate structure where both of the non-reducingtermini have a galactose. “G0-F” refers to a carbohydrate structurewhere there is no galactose at either of the two non-reducing terminiand there is no core fucosylation. Panel 1A: glycans isolated fromcontrol (untreated) h1F6 antibody. Panel 1B: glycans isolated from h1F6antibodies expressed in the presence of 50 μm alkynyl fucose peracetate.Panel 1C: glycans isolated from h1F6 antibodies expressed in thepresence of 50 μm alkynyl fucose peracetate and treated withβ-galactosidase to remove galactose from the G1 and G2 glycans.

FIG. 2 shows the results of effector function assays (ADCC) withantibodies produced from host cells cultured in the presence of alkynylfucose peracetate (AlkF). Specific lysis of control anti-CD70 antibody(shaded circles), anti-CD70 antibody from host cells cultured in thepresence of 50 μM and 100 μM AlkF (open circles and triangles,respectively) and nonbinding control IgG (shaded diamonds) wasdetermined by ⁵¹Cr release assay. CD70+786-O target cells were mixedwith NK-enriched PBMCs at an effector to target ratio of 10:1.

FIGS. 3A and 3B show the results of Fcγ receptor binding assays withcontrol anti-CD70 antibody and antibody from host cells cultured in thepresence of 50 μM alkynyl fucose peracetate (AlkF). The relativeaffinity for each receptor was determined by a competition binding assaybetween labeled parent antibody and increasing concentrations ofunlabeled parent (shaded squares) or non-core fucosylated (shadedtriangles) anti-CD70 mAb. FIG. 3A shows binding competition for humanFcγ receptor (CD16)-expressing cells. FIG. 3B shows binding competitionfor murine Fcγ receptor (CD16)-expressing cells.

FIGS. 4A, 4B, 4C and 4D show the results of LC-MS (Q-Tof) analysis offour antibodies cultured in the absence or presence of alkynyl fucoseperacetate (upper and lower portions, respectively, of each pair ofpanels). GO, G1 and G0-F are as indicated supra. “G1-F” refers to thecarbohydrate structure where one of the non-reducing termini has agalactose and there is no core fucosylation (a mixture of two isomers).FIG. 4A: anti-CD70 antibody. FIG. 4B: anti-CD19 antibody. FIG. 4C:anti-CD30 antibody. FIG. 4D: anti-CD33 antibody.

FIGS. 5A and 5 B show the results of effector function (ADCC) assays ofa humanized CD19 antibody cultured in the absence or presence of alkynylfucose peracetate (core fucosylated (squares) or non-core fucosylated(triangles), respectively) on NK cells having the 158V and 158Fphenotypes (panels A and B, respectively).

FIG. 6 shows the results of a titration of alkynyl fucose peracetate(“Alk Fuc peracetate”) on a culture of host cells expressing h1F6antibody and the effect on production of Ab with core fucosylation (G0).

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “antibody” refers to (a) immunoglobulin polypeptides andimmunologically active portions of immunoglobulin polypeptides, i.e.,polypeptides of the immunoglobulin family, or fragments thereof, thatcontain an antigen binding site that immunospecifically binds to aspecific antigen (e.g., CD70) and an Fc domain comprising a complexN-glycoside-linked sugar chain(s), or (b) conservatively substitutedderivatives of such immunoglobulin polypeptides or fragments thatimmunospecifically bind to the antigen (e.g., CD70). Antibodies aregenerally described in, for example, Harlow & Lane, Antibodies: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1988). Unlessotherwise apparent from the context, reference to an antibody alsoincludes antibody derivatives as described in more detail below.

An “antibody derivative” means an antibody, as defined above (includingan antibody fragment), or Fc domain or region of an antibody comprisinga complex N-glycoside linked sugar chain, that is modified by covalentattachment of a heterologous molecule such as, e.g., by attachment of aheterologous polypeptide (e.g., a ligand binding domain of heterologousprotein), or by glycosylation (other than core fucosylation),deglycosylation (other than non-core fucosylation), acetylation,phosphorylation or other modification not normally associated with theantibody or Fc domain or region.

The term “monoclonal antibody” refers to an antibody that is derivedfrom a single cell clone, including any eukaryotic or prokaryotic cellclone, or a phage clone, and not the method by which it is produced.Thus, the term “monoclonal antibody” is not limited to antibodiesproduced through hybridoma technology.

The term “Fc region” refers to the constant region of an antibody, e.g.,a C_(H)1-hinge-C_(H)2-C_(H)3 domain, optionally having a C_(H)4 domain,or a conservatively substituted derivative of such an Fc region.

The term “Fc domain” refers to the constant region domain of anantibody, e.g., a C_(H)1, hinge, C_(H)2, C_(H)3 or C_(H)4 domain, or aconservatively substituted derivative of such an Fc domain.

An “antigen” is a molecule to which an antibody specifically binds.

The terms “specific binding” and “specifically binds” mean that theantibody or antibody derivative will bind, in a highly selective manner,with its corresponding target antigen and not with the multitude ofother antigens. Typically, the antibody or antibody derivative bindswith an affinity of at least about 1×10⁻⁷ M, and preferably 10⁻⁸ M to10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M and binds to the predeterminedantigen with an affinity that is at least two-fold greater than itsaffinity for binding to a non-specific antigen (e.g., BSA, casein) otherthan the predetermined antigen or a closely-related antigen.

The terms “inhibit” or “inhibition of” means to reduce by a measurableamount, or to prevent entirely.

As used herein, “alkynyl fucose peracetate” refers to any or all formsof alkynyl fucose (5-ethynylarabinose) with acetate groups on positionsR¹⁻⁴ (see formula I and II, infra), including6-ethynyl-tetrahydro-2H-pyran-2,3,4,5-tetrayl tetraacetate, includingthe (2S,3S,4R,5R,6S) and (2R,3S,4R,5R,6S) isomers, and5-((S)-1-hydroxyprop-2-ynyl)-tetrahydrofuran-2,3,4-triyl tetraacetate,including the (2S,3S,4R,5R) and (2R,3S,4R,5R) isomers, and the aldoseform, unless otherwise indicated by context. The terms “alkynyl fucosetriacetate”, “alkynyl fucose diacetate” and “alkynyl fucose monoacetate”refer to the indicated tri-, di- and mono-acetate forms of alkynylfucose, respectively.

Unless otherwise indicated by context, the term “alkyl” refers to asubstituted or unsubstituted saturated straight or branched hydrocarbonhaving from 1 to 20 carbon atoms (and all combinations andsubcombinations of ranges and specific numbers of carbon atoms therein),with from 1 to 3, 1 to 8 or 1 to 10 carbon atoms being preferred.Examples of alkyl groups are methyl, ethyl, n-propyl, iso-propyl,n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl,2-methyl-2-butyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl,3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl,3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl,3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, and3,3-dimethyl-2-butyl.

Alkyl groups, whether alone or as part of another group, can beoptionally substituted with one or more groups, preferably 1 to 3 groups(and any additional substituents selected from halogen), including, butnot limited to: halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH, ═O, —NH₂,—NH(R′), —N(R′)₂ and —CN; where each R′ is independently selected from—H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or aryl. In someembodiments, the —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), aryl, and R′ groups can be further substituted. Such furthersubstituents include, for example, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈alkynyl, halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), -aryl, —C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂, —C(O)NHR″,—C(O)N(R″)₂, —NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH, —NH₂,—NH(R″), —N(R″)₂ and —CN, where each R″ is independently selected fromH, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or aryl wherein saidfurther substituents are preferably unsubstituted. In some embodiments,the —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), aryl, andR′ groups are not further substituted.

Unless otherwise indicated by context, the terms “alkenyl” and “alkynyl”refer to substituted or unsubstituted straight and branched carbonchains having from 2 to 20 carbon atoms (and all combinations andsubcombinations of ranges and specific numbers of carbon atoms therein),with from 2 to 3, 2 to 4, 2 to 8 or 2 to 10 carbon atoms beingpreferred. An alkenyl chain has at least one double bond in the chainand an alkynyl chain has at least one triple bond in the chain. Examplesof alkenyl groups include, but are not limited to, ethylene or vinyl,allyl, -1 butenyl, -2 butenyl, -isobutylenyl, -1 pentenyl, -2 pentenyl,3-methyl-1-butenyl, -2 methyl 2 butenyl, and -2,3 dimethyl 2 butenyl.Examples of alkynyl groups include, but are not limited to, acetylenic,propargyl, acetylenyl, propynyl, -1 butynyl, -2 butynyl, -1 pentynyl, -2pentynyl, and -3 methyl 1 butynyl.

Alkenyl and alkynyl groups, whether alone or as part of another group,can be optionally substituted with one or more groups, preferably 1 to 3groups (and any additional substituents selected from halogen),including but not limited to: halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈alkenyl), —O—(C₂-C₈ alkynyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′,—C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′,—S(O)R′, —OH, ═O, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from H, —C₁-C₈ alkyl, —C₂—C alkenyl, —C₂-C₈alkynyl, or aryl. In some embodiments, the —O—(C₁-C₈ alkyl), —O—(C₂-C₈alkenyl), —O—(C₂-C₈ alkynyl), aryl, and R′ groups can be furthersubstituted. Such further substituents include, for example, —C₁-C₈alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, halogen, —O—(C₁-C₈ alkyl),—O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), -aryl, —C(O)R″, —OC(O)R″,—C(O)OR″, —C(O)NH₂, —C(O)NHR″, —C(O)N(R″)₂, —NHC(O)R″, —SR″, —SO₃R″,—S(O)₂R″, —S(O)R″, —OH, —NH₂, —NH(R″), —N(R″)₂ and —CN, where each R″ isindependently selected from H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈alkynyl, or aryl, wherein said further substituents are preferablyunsubstituted. In some embodiments, the —O—(C₁-C₇ alkyl), —O—(C₂-C₈alkenyl), —O—(C₂-C₈ alkynyl), -aryl, and R′ groups are not furthersubstituted.

Unless otherwise indicated by context, the term “alkylene” refers to asubstituted or unsubstituted saturated branched or straight chainhydrocarbon radical having from 1 to 20 carbon atoms (and allcombinations and subcombinations of ranges and specific numbers ofcarbon atoms therein), with from 1 to 8 or 1 to 10 carbon atoms beingpreferred and having two monovalent radical centers derived by theremoval of two hydrogen atoms from the same or two different carbonatoms of a parent alkane. Typical alkylenes include, but are not limitedto, methylene, ethylene, propylene, butylene, pentylene, hexylene,heptylene, ocytylene, nonylene, decalene, 1,4-cyclohexylene, and thelike.

Alkylene groups, whether alone or as part of another group, can beoptionally substituted with one or more groups, preferably 1 to 3 groups(and any additional substituents selected from halogen), including, butnot limited to: halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)2_(R)′, —S(O)R′, —OH, ═O,—NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ is independently selectedfrom H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryl. In someembodiments, the —O—(C₁-C₈ alkyl), —O—(C₂-C8 alkenyl), —O—(C₂-C₈alkynyl), aryl, and R′ groups can be further substituted. Such furthersubstituents include, for example, C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈alkynyl, halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), -aryl, —C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂, —C(O)NHR″,—C(O)N(R″)₂, —NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH, —NH₂,—NH(R″), —N(R″)₂ and —CN, where each R″ is independently selected fromH, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or aryl wherein saidfurther substituents are preferably unsubstituted. In some embodiments,the —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), -aryl, andR′ groups are not further substituted.

Unless otherwise indicated by context, the term “aryl” refers to asubstituted or unsubstituted monovalent aromatic hydrocarbon radical of6-20 carbon atoms (and all combinations and subcombinations of rangesand specific numbers of carbon atoms therein) derived by the removal ofone hydrogen atom from a single carbon atom of a parent aromatic ringsystem. Some aryl groups are represented in the exemplary structures as“Ar”. Typical aryl groups include, but are not limited to, radicalsderived from benzene, substituted benzene, phenyl, naphthalene,anthracene, biphenyl, and the like.

An aryl group, whether alone or as part of another group, can beoptionally substituted with one or more, preferably 1 to 5, or even 1 to2 groups including, but not limited to: halogen, —C₁-C₈ alkyl, —C₂-C₈alkenyl, —C₂-C₈ alkynyl, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH, —NO₂,—NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ is independently selectedfrom H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or aryl. In someembodiments, the C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, —O—(C₁-C₈alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), aryl and R′ groups canbe further substituted. Such further substituents include, for example,—C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, halogen, —O—(C₁-C₈ alkyl),—O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), -aryl, —C(O)R″, —OC(O)R″,—C(O)OR″, —C(O)NH₂, —C(O)NHR″, —C(O)N(R″)₂, —NHC(O)R″, —SR″, —SO₃R″,—S(O)₂R″, —S(O)R″, —OH, —NH₂, —NH(R″), —N(R″)₂ and —CN, where each R″ isindependently selected from —H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈alkynyl, or aryl wherein said further substituents are preferablyunsubstituted. In some embodiments, the —C₁-C₈ alkyl, —C₂-C₈ alkenyl,—C₂-C₈ alkynyl, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), aryl and R′ groups are not further substituted.

Unless otherwise indicated by context, the term “heterocycle” refers toa substituted or unsubstituted monocyclic ring system having from 3 to7, or 3 to 10, ring atoms (also referred to as ring members) wherein atleast one ring atom is a heteroatom selected from N, O, P, or S (and allcombinations and subcombinations of ranges and specific numbers ofcarbon atoms and heteroatoms therein). The heterocycle can have from 1to 4 ring heteroatoms independently selected from N, O, P, or S. One ormore N, C, or S atoms in a heterocycle can be oxidized. A monocylicheterocycle preferably has 3 to 7 ring members (e.g., 2 to 6 carbonatoms and 1 to 3 heteroatoms independently selected from N, O, P, or S).The ring that includes the heteroatom can be aromatic or non-aromatic.Unless otherwise noted, the heterocycle is attached to its pendant groupat any heteroatom or carbon atom that results in a stable structure.

Heterocycles are described in Paquette, “Principles of ModernHeterocyclic Chemistry” (W. A. Benjamin, New York, 1968), particularlyChapters 1, 3, 4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds,A series of Monographs” (John Wiley & Sons, New York, 1950 to present),in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc.82:5566 (1960).

Examples of “heterocycle” groups include by way of example and notlimitation pyridyl, dihydropyridyl, tetrahydropyridyl (piperidyl),thiazolyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl,imidazolyl, tetrazolyl, fucosyl, azirdinyl, azetidinyl, oxiranyl,oxetanyl, and tetrahydrofuranyl.

A heterocycle group, whether alone or as part of another group, can beoptionally substituted with one or more groups, preferably 1 to 2groups, including but not limited to: —C₁-C₈ alkyl, —C₂-C₈ alkenyl,—C₂-C₈ alkynyl, halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH, —NH₂,—NH(R′), —N(R′)₂ and —CN; where each R′ is independently selected fromH, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or -aryl. In someembodiments, the O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, aryl, and R′groups can be further substituted. Such further substituents include,for example, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, halogen,—O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), -aryl,—C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂, —C(O)NHR″, —C(O)N(R″)₂,—NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH, —NH₂, —NH(R″), —N(R″)₂and —CN, where each R″ is independently selected from H, —C₁-C₈ alkyl,—C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or aryl wherein said furthersubstituents are preferably unsubstituted. In some embodiments, the—O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), —C₁-C₈ alkyl,—C₂-C₈ alkenyl, —C₂-C₈ alkynyl, aryl, and R′ groups are not substituted.

By way of example and not limitation, carbon-bonded heterocycles can bebonded at the following positions: position 2, 3, 4, 5, or 6 of apyridine; position 3, 4, 5, or 6 of a pyridazine; position 2, 4, 5, or 6of a pyrimidine; position 2, 3, 5, or 6 of a pyrazine; position 2, 3, 4,or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole ortetrahydropyrrole; position 2, 4, or 5 of an oxazole, imidazole orthiazole; position 3, 4, or 5 of an isoxazole, pyrazole, or isothiazole;position 2 or 3 of an aziridine; position 2, 3, or 4 of an azetidine.Exemplary carbon bonded heterocycles can include 2-pyridyl, 3-pyridyl,4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl,5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl,5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl,6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

By way of example and not limitation, nitrogen bonded heterocycles canbe bonded at position 1 of an aziridine, azetidine, pyrrole,pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine,2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline,3-pyrazoline, piperidine, piperazine, indole, indoline, or 1H-indazole;position 2 of a isoindole, or isoindoline; and position 4 of amorpholine. Still more typically, nitrogen bonded heterocycles include1-aziridyl, 1-azetidyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and1-piperidinyl.

Unless otherwise noted, the term “carbocycle,” refers to a substitutedor unsubstituted, saturated or unsaturated non-aromatic monocyclic ringsystem having from 3 to 6 ring atoms (and all combinations andsubcombinations of ranges and specific numbers of carbon atoms therein)wherein all of the ring atoms are carbon atoms.

Carbocycle groups, whether alone or as part of another group, can beoptionally substituted with, for example, one or more groups, preferably1 or 2 groups (and any additional substituents selected from halogen),including, but not limited to: halogen, C₁-C₈ alkyl, —C₂-C₈ alkenyl,—C₂-C₈ alkynyl, —O—(C₂-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH, ═O, —NH₂,—NH(R′), —N(R′)₂ and —CN; where each R′ is independently selected fromH, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or aryl. In someembodiments, the —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, —O—(C₁-C₈alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), -aryl and R′ groups canbe further substituted. Such further substituents include, for example,—C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, halogen, —O—(C₁-C₈ alkyl),—O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), -aryl, —C(O)R″, —OC(O)R″,—C(O)OR″, —C(O)NH₂, —C(O)NHR″, —C(O)N(R″)₂, —NHC(O)R″, —SR″, —SO₃R″,—S(O)₂R″, —S(O)R″, —OH, —NH₂, —NH(R″), —N(R″)₂ and —CN, where each R″ isindependently selected from H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈alkynyl, or aryl wherein said further substituents are preferablyunsubstituted. In some embodiments, the —C₁-C₈ alkyl, —C₂-C₈ alkenyl,—C₂-C₈ alkynyl, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), aryl and R′ groups are not substituted.

Examples of monocyclic carbocylic substituents include cyclopropyl,cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl,1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl,1-cyclohex-3-enyl, cycloheptyl, cyclooctyl, -1,3-cyclohexadienyl,-1,4-cyclohexadienyl, -1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl,and -cyclooctadienyl.

When any variable occurs more than one time in any constituent or in anyformula, its definition in each occurrence is independent of itsdefinition at every other. Combinations of substituents and/or variablesare permissible only if such combinations result in stable compounds.

Unless otherwise indicated by context, a hyphen (-) designates the pointof attachment to the pendant molecule. Accordingly, the term “—(C₁-C₁₀alkylene)aryl” or “—C₁-C₁₀ alkylene(aryl)” refers to a C₁-C₁₀ alkyleneradical as defined herein wherein the alkylene radical is attached tothe pendant molecule at any of the carbon atoms of the alkylene radicaland one of the hydrogen atom bonded to a carbon atom of the alkyleneradical is replaced with an aryl radical as defined herein.

When a particular group is “substituted”, that group may have one ormore substituents, preferably from one to five substituents, morepreferably from one to three substituents, most preferably from one totwo substituents, independently selected from the list of substituents.The group can, however, generally have any number of substituentsselected from halogen.

It is intended that the definition of any substituent or variable at aparticular location in a molecule be independent of its definitionselsewhere in that molecule. It is understood that substituents andsubstitution patterns on the compounds of this invention can be selectedby one of ordinary skill in the art to provide compounds that arechemically stable and that can be readily synthesized by techniquesknown in the art as well as those methods set forth herein.

The term “pharmaceutically acceptable” means approved by a regulatoryagency of the Federal or a state government or listed in the U.S.Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “pharmaceuticallycompatible ingredient” refers to a pharmaceutically acceptable diluent,adjuvant, excipient, or vehicle with which the antibody or antibodyderivative is administered.

The term “biologically acceptable” means suitable for use in the cultureof cell lines for the manufacture of antibodies. Exemplary biologicallyacceptable salts include, but are not limited, to sulfate, citrate,acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate,phosphate, acid phosphate, isonicotinate, lactate, salicylate, acidcitrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucuronate,saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate(i.e., 1,1′-methylene bis-(2 hydroxy 3-naphthoate)) salts. Abiologically acceptable salt may involve the inclusion of anothermolecule such as an acetate ion, a succinate ion or other counterion.The counterion may be any organic or inorganic moiety that stabilizesthe charge on the parent compound. Furthermore, a biologicallyacceptable salt may have more than one charged atom in its structure.Instances where multiple charged atoms are part of the biologicallyacceptable salt can have multiple counter ions. Hence, a biologicallysalt can have one or more charged atoms and/or one or more counterion.

A “biologically acceptable solvate” or “solvate” refer to an associationof one or more solvent molecules and a fucose analog. Examples ofsolvents that form biologically acceptable solvates include, but are notlimited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate,acetic acid, and ethanolamine.

“Small electron-withdrawing groups” refers to any substituent that hasgreater electronegativity at the site of substituent attachment than,e.g., a hydrogen atom or hydroxy group or relative to the substituentpresent in fucose at that site. Generally, the smallelectron-withdrawing group has 10 or fewer atoms (other than hydrogen)and includes groups such as nitro; cyano and cyanoalkyl (e.g.,—CH₂CH₂CN); halogen; acetylene or other alkynes or halo alkynes (e.g.,—C≡CCF₃); alkenes or halo alkenes; allenes; carboxylic acids, ester,amides and halo substituted forms thereof; sulfonic and phosphonicacids, esters and amides, and halo substituted forms thereof; haloalkylgroups (e.g., —CF₃, —CHF₂, —CH₂CF₃), acyl and haloacyl groups (e.g.,—C(O)CH₃ and —C(O)CF₃); alkylsulfonyl and haloalkylsulfonyl (e.g.,—S(O)₂alkyl and —S(O)₂haloalkyl); aryloxy (e.g, phenoxy and substitutedphenoxy); aralkyloxy (e.g, benzyloxy and substituted benzyloxy); andoxiranes. Preferred small electron-withdrawing groups are those having8, 7 or 6 or fewer atoms (other than hydrogen).

Therapeutic agents of the invention are typically substantially purefrom undesired contaminant. This means that an agent is typically atleast about 50% w/w (weight/weight) purity, as well as beingsubstantially free from interfering proteins and contaminants. Sometimesthe agents are at least about 80% w/w and, more preferably at least 90%or about 95% w/w purity. Using conventional protein purificationtechniques, homogeneous peptides of at least 99% w/w can be obtained.

General

The invention provides compositions and methods for preparing antibodiesand antibody derivatives with reduced core fucosylation. The methods arepremised in part on the unexpected results presented in the Examplesshowing that culturing host cells, expressing an antibody or antibodyderivative of interest, in culture media comprising a fucose analogproduces an antibody or antibody derivative having reduced corefucosylation. As used herein, “core fucosylation” refers to addition offucose (“fucosylation”) to N-acetylglucosamine (“GlcNAc”) at thereducing terminal of an N-linked glycan. Also provided are antibodiesand antibody derivatives produced by such methods. In other aspects,fucose analogs and culture media comprising an effective amount of afucose analog(s) are provided.

In some embodiments, fucosylation of complex N-glycoside-linked sugarchains bound to the Fc region (or domain) is reduced. As used herein, a“complex N-glycoside-linked sugar chain” is typically bound toasparagine 297 (according to the number of Kabat), although a complexN-glycoside linked sugar chain can also be linked to other asparagineresidues. As used herein, the complex N-glycoside-linked sugar chain hasa bianntennary composite sugar chain, mainly having the followingstructure:

where ± indicates the sugar molecule can be present or absent, and thenumbers indicate the position of linkages between the sugar molecules.In the above structure, the sugar chain terminal which binds toasparagine is called a reducing terminal (at right), and the oppositeside is called a non-reducing terminal. Fucose is usually bound toN-acetylglucosamine (“GlcNAc”) of the reducing terminal, typically by anα1,6 bond (the 6-position of GlcNAc is linked to the 1-position offucose). “Gal” refers to galactose, and “Man” refers to mannose.

A “complex N-glycoside-linked sugar chain” excludes a high mannose typeof sugar chain, in which only mannose is incorporated at thenon-reducing terminal of the core structure, but includes 1) a complextype, in which the non-reducing terminal side of the core structure hasone or more branches of galactose-N-acetylglucosamine (also referred toas “gal-GlcNAc”) and the non-reducing terminal side of Gal-GlcNAcoptionally has a sialic acid, bisecting N-acetylglucosamine or the like;or 2) a hybrid type, in which the non-reducing terminal side of the corestructure has both branches of the high mannose N-glycoside-linked sugarchain and complex N-glycoside-linked sugar chain.

In some embodiments, the “complex N-glycoside-linked sugar chain”includes a complex type in which the non-reducing terminal side of thecore structure has zero, one or more branches ofgalactose-N-acetylglucosamine (also referred to as “gal-GlcNAc”) and thenon-reducing terminal side of Gal-GlcNAc optionally further has astructure such as a sialic acid, bisecting N-acetylglucosamine or thelike.

According to the present methods, typically only a minor amount offucose is incorporated into the complex N-glycoside-linked sugarchain(s). For example, in various embodiments, less than about 60%, lessthan about 50%, less than about 40%, less than about 30%, less thanabout 20%, less than about 15%, less than about 10%, less than about 5%,or less than about 1% of the antibody or antibody derivative has corefucosylation by fucose. In some embodiments, substantially none (i.e.,less than 0.5%) of the antibody or antibody derivative has corefucosylation by fucose.

In certain embodiments, only a minor amount of a fucose analog (or ametabolite or product of the fucose analog) is incorporated into thecomplex N-glycoside-linked sugar chain(s). For example, in variousembodiments, less than about 40%, less than about 30%, less than about20%, less than about 15%, less than about 10%, less than about 5%, orless than about 1% of the antibody or antibody derivative has corefucosylation by a fucose analog or a metabolite or product of the fucoseanalog. In some embodiments, substantially none (i.e., less than 0.5%)of the antibody or antibody derivative has core fucosylation by a fucoseanalog or a metabolite or product of the fucose analog.

Fucose Analogs

In one aspect, fucose analogs are described that reduce theincorporation of fucose into complex N-glycoside-linked sugar chains ofantibodies or antibody derivatives produced by host cells. Suitablefucose analogs (identified below as Formula I, II, III, IV, V and VI)are those that can be added to the host cell culture media and thatinhibit core fucosylation of complex N-glycoside-linked sugar chains ofantibodies or antibody derivatives. The fucose analog is typically takenup by host cells (e.g., by active transport or passive diffusion).

In some embodiments, a fucose analog (or an intracellular metabolite orproduct of the fucose analog) inhibits an enzyme(s) in the fucosesalvage pathway. (As used herein, an intracellular metabolite can be,for example, a GDP-modified analog or a fully or partially de-esterifiedanalog. A product can be, for example, a fully or partiallyde-esterified analog.) For example, a fucose analog (or an intracellularmetabolite or product of the fucose analog) can inhibit the activity offucokinase, or GDP-fucose-pyrophosphorylase. In some embodiments, afucose analog (or an intracellular metabolite or product of the fucoseanalog) inhibits fucosyltransferase (preferably a1,6-fucosyltransferase, e.g., the FUT8 protein). In some embodiments, afucose analog (or an intracellular metabolite or product of the fucoseanalog) can inhibit the activity of an enzyme in the de novo syntheticpathway for fucose. For example, a fucose analog (or an intracellularmetabolite or product of the fucose analog) can inhibit the activity ofGDP-mannose 4,6-dehydratase or/or GDP-fucose synthetase. In someembodiments, the fucose analog (or an intracellular metabolite orproduct of the fucose analog) can inhibit a fucose transporter (e.g.,GDP-fucose transporter).

In some embodiments, the fucose analog has the following formula (I) or(II):

or a biologically acceptable salt or solvate of the analog, wherein eachof formula (I) or (II) can be the alpha or beta anomer or thecorresponding aldose form. In the above formulae, each of R¹-R⁴ isindependently selected from the group consisting of —OH, —OC(O)H,—OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀ alkenyl, —OC(O)C₂-C₁₀ alkynyl,—OC(O)aryl, —OC(O)heterocycle, —OC(O)C₁-C₁₀ alkylene aryl, —OC(O)C₂-C₁₀alkenylene aryl, —OC(O)C₂-C₁₀ alkynylene aryl, —OC(O)C₂-C₁₀ alkyleneheterocycle, —OC(O)C₂-C₁₀ alkenylene heterocycle, —OC(O)C₂-C₁₀alkynylene heterocycle, —OC(O)CH₂O(CH₂CH₂O)_(n)CH₃,—OC(O)CH₂CH₂O(CH₂CH₂O)_(n)CH₃, —O-tri-C₁-C₃ alkyl silyl, —OC₁—C₁₀ alkyl,—OCH₂OC(O) alkyl, —OCH₂OC(O) alkenyl, —OCH₂OC(O) alkynyl, —OCH₂OC(O)aryl, —OCH₂OC(O) heterocycle, —OCH₂OC(O)O alkyl, —OCH₂OC(O)O alkenyl,—OCH₂OC(O)O alkynyl, —OCH₂OC(O)O aryl and —OCH₂OC(O)O heterocycle,wherein each n is an integer independently selected from 0-5; and R⁵ isselected from the group consisting of —C≡CH, —C≡CCH₃, —CH₂C≡CH,—C(O)OCH₃, —CH(OAc)CH₃, —CN, —CH₂CN, —CH₂X (wherein X is Br, Cl or I),and methoxiran.

In some embodiments, the fucose analog has formula (I) or (II), wherein:each of R¹-R⁴ is independently selected from the group consisting of—OH, —OC(O)H, —OC(O)C₁-C₁₀ alkyl, —OC(O)aryl, —OC(O)heterocycle,—OC(O)C₁-C₁₀ alkylene aryl, —OC(O)C₁-C₁₀ alkylene heterocycle,—OC(O)CH₂O(CH₂CH₂O)CH₃, —OC(O)CH₂CH₂O(CH₂CH₂O)_(n)CH₃, —O-tri-C₁-C₃silyl, —OC₂—C₁₀ alkyl, —OCH₂OC(O) alkyl, —OCH₂OC(O)O alkyl, —OCH₂OC(O)aryl, and —OCH₂OC(O)O aryl, wherein each n is an integer independentlyselected from 0-5; and R⁵ is selected from the group consisting of—C≡CH, —C≡CCH₃, —CH₂C≡CH, —C(O)OCH₃, —CH(OAc)CH₃, —CN, —CH₂CN, —CH₂X(wherein X is Br, Cl or I), and methoxiran.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, —OC(O)H, —OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀ alkenyl, —OC(O)C₂-C₁₀alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C₁-C₁₀ alkylene aryl,—OC(O)C₂-C₁₀ alkenylene aryl, —OC(O)C₂-C₁₀ alkynylene aryl, —OC(O)C₂-C₁₀alkylene heterocycle, —OC(O)C₂-C₁₀ alkenylene heterocycle, and—OC(O)C₂-C₁₀ alkynylene heterocycle; and R⁵ is selected from the groupconsisting of —C≡CH, —C≡CCH₃, —CH₂C≡CH, —C(O)OCH₃, —CH(OAc)CH₃, —CN,—CH₂CN, —CH₂X (wherein X is Br, Cl or I), and methoxiran.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—O-tri-C₁-C₃ silyl and —OC₁—C₁₀ alkyl; and R⁵ is selected from the groupconsisting of —C⬇CH, —C≡CCH₃, —CH₂C≡CH, —C(O)OCH₃, —CH(OAc)CH₃, —CN,—CH₂CN, —CH₂X (wherein X is Br, Cl or I), and methoxiran.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OCH₂OC(O) alkyl, —OCH₂OC(O) alkenyl, —OCH₂OC(O) alkynyl, —OCH₂OC(O)aryl, —OCH₂OC(O) heterocycle, —OCH₂OC(O)O alkyl, —OCH₂OC(O)O alkenyl,—OCH₂OC(O)O alkynyl, —OCH₂OC(O)O aryl, and —OCH₂OC(O)O heterocycle; andR⁵ is selected from the group consisting of —C≡CH, —C≡CCH₃, —CH₂C≡CH,—C(O)OCH₃, —CH(OAc)CH₃, —CN, —CH₂CN, —CH₂X (wherein X is Br, Cl or I),and methoxiran.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, —OC(O)H, —OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀ alkenyl, —OC(O)C₂-C₁₀alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C₁-C₁₀ alkylene aryl,—OC(O)C₂-C₁₀ alkenylene aryl, —OC(O)C₂-C₁₀ alkynylene aryl, —OC(O)C₂-C₁₀alkylene heterocycle, —OC(O)C₂-C₁₀ alkenylene heterocycle, and—OC(O)C₂-C₁₀ alkynylene heterocycle; and R⁵ is selected from the groupconsisting of —C≡CH, —C≡CCH₃, —CH₂C≡CH, —C(O)OCH₃, —CH(OAc)CH₃, —CN,—CH₂CN, and methoxiran.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, —OC(O)H, —OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀ alkenyl, —OC(O)C₂-C₁₀alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C₁-C₁₀ alkylene aryl,—OC(O)C₂-C₁₀ alkenylene aryl, —OC(O)C₂-C₁₀ alkynylene aryl, —OC(O)C₂-C₁₀alkylene heterocycle, —OC(O)C₂-C₁₀ alkenylene heterocycle, and—OC(O)C₂-C₁₀ alkynylene heterocycle; and R⁵ is selected from the groupconsisting of —CH₂I, —CH₂Br, and —CH₂Cl.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, —OC(O)H, —OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀ alkenyl, —OC(O)C₂-C₁₀alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C₁-C₁₀ alkylene aryl,—OC(O)C₂-C₁₀ alkenylene aryl, —OC(O)C₂-C₁₀ alkynylene aryl, —OC(O)C₂-C₁₀alkylene heterocycle, —OC(O)C₂-C₁₀ alkenylene heterocycle, and—OC(O)C₂-C₁₀ alkynylene heterocycle; and R⁵ is selected from the groupconsisting of —C≡CH, —C≡CCH₃ and —CH₂C≡CH.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, —OC(O)H, —OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀ alkenyl, —OC(O)C₂-C₁₀alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C₁-C₁₀ alkylene aryl,—OC(O)C₂-C₁₀ alkenylene aryl, —OC(O)C₂-C₁₀ alkynylene aryl, —OC(O)C₂-C₁₀alkylene heterocycle, —OC(O)C₂-C₁₀ alkenylene heterocycle, and—OC(O)C₂-C₁₀ alkynylene heterocycle; and R⁵ is selected from the groupconsisting of —C≡CH, —C≡CCH₃, —(CH₂)_(n)(CN) (where n=0 or 1) and—CO(O)CH₃.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, —OC(O)H, —OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀ alkenyl, —OC(O)C₂-C₁₀alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C₁-C₁₀ alkylene aryl,—OC(O)C₂-C₁₀ alkenylene aryl, —OC(O)C₂-C₁₀ alkynylene aryl, —OC(O)C₂-C₁₀alkylene heterocycle, —OC(O)C₂-C₁₀ alkenylene heterocycle, and—OC(O)C₂-C₁₀ alkynylene heterocycle; and R⁵ is selected from the groupconsisting of —C≡CH, —C≡CCH₃, —CH₂CN and —CO(O)CH₃.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, —OC(O)H, —OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀ alkenyl, —OC(O)C₂-C₁₀alkynyl, —OC(O)aryl, —OC(O)heterocycle, —OC(O)C₁-C₁₀ alkylene aryl,—OC(O)C₂-C₁₀ alkenylene aryl, —OC(O)C₂-C₁₀ alkynylene aryl, —OC(O)C₂-C₁₀alkylene heterocycle, —OC(O)C₂-C₁₀ alkenylene heterocycle, and—OC(O)C₂-C₁₀ alkynylene heterocycle; and R⁵ is selected from the groupconsisting of —C≡CH, —C≡CCH₃, —CH(OAc)CH₃, —CH₂CN, and —CO(O)CH₃.

In some embodiments, the fucose analog has formula (I) or (II), whereinR⁵ is as defined herein, and each of R¹-R⁴ is other than hydroxyl.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is independently selected from the group consisting of—OH, and —OAc; and R⁵ is selected from the group consisting of —C≡CH,—C≡CCH₃, —CH(OAc)CH₃, —CH₂CN, and —CO(O)CH₃.

In some embodiments, the fucose analog has formula (I) or (II), whereineach of R¹-R⁴ is —OH or an ester selected from the group consisting of—OC(O)H, —OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀ alkenyl, —OC(O)C₂-C₁₀ alkynyl,—OC(O)aryl, —OC(O)heterocycle, —OC(O)C₁-C₁₀ alkylene aryl, —OC(O)C₂-C₁₀alkenylene aryl, —OC(O)C₂-C₁₀ alkynylene aryl, —OC(O)C₁-C₁₀ alkyleneheterocycle, —OC(O)C₂-C₁₀ alkenylene heterocycle, —OC(O)C₂-C₁₀alkynylene heterocycle, —OC(O)CH₂O(CH₂CH₂O)_(n)CH₃ (where n is 0-5), and—OC(O)CH₂CH₂O(CH₂CH₂O)_(n)CH₃ (where n is 0-5); and R⁵ is selected fromthe group consisting of —C≡CH, —C≡CCH₃, —CH₂C≡CH, —C(O)OCH₃,—CH(OAc)CH₃, —CN, —CH₂CN, —CH₂X (wherein X is Br, Cl or I), andmethoxiran.

In some embodiments, the fucose analog has a molecular weight of lessthan 2000 daltons. In some embodiments, the fucose analog has amolecular weight of less than 1000 daltons.

In some embodiments, R⁵ is not substituted.

In some embodiments, each of R¹-R⁴ is not substituted.

In some embodiments, R⁵ is not a ketone (—C(O)alkyl).

In some embodiments, R⁵ is not —CHCH₃OAc.

In some embodiments, R⁵ is not —CHCH₃OAc, when each of R¹-R⁴ is —OAc.

In some embodiments, R⁵ is not —C≡CH₃.

In some embodiments, R⁵ is not —C≡CH₃, when any of R¹-R⁴ is —OAc.

In some embodiments, R⁵ is not —C≡CH₃, when any of R¹-R⁴ is —OC(O)alkyl.

In some embodiments, R⁵ is not —C≡CH₃, when each of R¹-R⁴ is—OC(O)alkyl.

In some embodiments, R⁵ is not —C≡CH₃, when each of R¹-R⁴ is OH.

In some embodiments, the fucose analog is alkynyl fucose peracetate. Insome embodiments, the fucose analog is alkynyl fucose triacetate. Insome embodiments, the fucose analog is alkynyl fucose diacetate. In someembodiments, the fucose analog is mixture of alkynyl fucose peracetate,alkynyl fucose triacetate and alkynyl fucose diacetate.

In some embodiments, the fucose analog is mixture of alkynyl fucoseperacetate, alkynyl fucose triacetate, alkynyl fucose diacetate andalkynyl fucose monoacetate.

In any of the various embodiments, the fucose analog is not fucose. Insome embodiments, the fucose analog is not alkynyl fucose peracetate. Insome embodiments, the fucose analog is not galactose or L-galactose.

In another group of embodiments, the fucose analog has the followingformula (III) or (IV):

or a biologically acceptable salt or solvate thereof, wherein each offormula (III) or (IV) can be the alpha or beta anomer or thecorresponding aldose form; and wherein,

-   each of R¹-R⁴ is independently selected from the group consisting of    fluoro, chloro, —OH, —OC(O)H, —OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀    alkenyl, —OC(O)C₂-C₁₀ alkynyl, —OC(O)aryl, —OC(O)heterocycle,    —OC(O)C₁-C₁₀ alkylene(aryl), —OC(O)C₂-C₁₀ alkylene(aryl),    —OC(O)C₂-C₁₀ alkynyl(aryl), —OC(O)C₁-C₁₀ alkylene heterocycle,    —OC(O)C₂-C₁₀ alkenylene(heterocycle), —OC(O)C₂-C₁₀ alkynyl    heterocycle, —OCH₂OC(O) alkyl, —OCH₂OC(O)O alkyl; —OCH₂OC(O) aryl,    —OCH₂OC(O)O aryl, —OC(O)CH₂O(CH₂CH₂O)_(n)CH₃,    —OC(O)CH₂CH₂O(CH₂CH₂O)_(n)CH₃, —O-tri-C₁-C₃ alkylsilyl and —OC₁—C₁₀    alkyl, wherein each n is an integer independently selected from 0-5;    and-   each of R^(2a) and R^(3a) is independently selected from the group    consisting of H, F and Cl;-   R⁵ is selected from the group consisting of —CH₃, —CHF₂, —CH═C≡CH₂,    —C≡CH, —C≡CCH₃, —CH₂C≡CH, —C(O)OCH₃, —CH(OAc)CH₃, —CN, —CH₂CN, —CH₂X    (wherein X is Br, Cl or I), and methoxiran;    wherein when R⁵ is other than —CH═C═CH₂ or —CHF₂, at least one of    R¹, R², R³, R^(2a) and R^(3a) is fluoro or chloro.

In some embodiments of formulae (III) or (IV), R¹ is F.

In some embodiments of formulae (III) or (IV), R² is F.

In some embodiments of formulae (III) or (IV), R³ is F.

In some embodiments of formulae (III) or (IV), R¹ and R² are each F.

In some embodiments of formulae (III) or (IV), R² and R^(2a) are each F.

In some embodiments of formulae (III) or (IV), R¹, R³ and R⁴ are eachindependently selected from —OH and —OAc; R² is F; and R⁵ is —CH₃.

In some embodiments of formulae (III) or (IV), R¹, R³ and R⁴ are eachindependently selected from —OH and —OAc; R² is F; R^(2a) and R^(3a) areeach H; and R⁵ is —CH₃.

In another group of embodiments, the fucose analog has the followingformula (V) or (VI):

or a biologically acceptable salt or solvate thereof, wherein each offormula (V) or (VI) can be the alpha or beta anomer or the correspondingaldose form; and wherein,each of R¹, R², R^(2a), R³, R^(3a) and R⁴ is independently selected fromthe group consisting of —OH, —OC(O)H, —OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀alkenyl, —OC(O)C₂-C₁₀ alkynyl, —OC(O)aryl, —OC(O)heterocycle,—OC(O)C₁-C₁₀ alkylene(aryl), —OC(O)C₂-C₁₀ alkylene(aryl), —OC(O)C₂-C₁₀alkynyl(aryl), —OC(O)C₁-C₁₀ alkylene heterocycle, —OC(O)C₂-C₁₀alkenylene(heterocycle), —OC(O)C₂-C₁₀ alkynyl heterocycle, —OCH₂OC(O)alkyl, —OCH₂OC(O)O alkyl, —OCH₂OC(O) aryl, —OCH₂OC(O)O aryl,—OC(O)CH₂O(CH₂CH₂O)₁CH₃, —OC(O)CH₂CH₂O(CH₂CH₂O)CH₃, —O-tri-C₁-C₃alkylsilyl, —OC₁—C₁₀ alkyl, and a small electron withdrawing group,wherein each n is an integer independently selected from 0-5;R⁵ is a member selected from the group consisting of —CH₃, —CH₂X,—CH(X′)—C₁-C₄ alkyl unsubstituted or substituted with halogen,—CH(X′)—C₂-C₄ alkene unsubstituted or substituted with halogen,—CH(X′)—C₂-C₄ alkyne unsubstituted or substituted with halogen,—CH═C(R¹⁰)(R¹¹), —C(CH₃)═C(R¹²)(R¹³), —C(R¹⁴)═C═C(R¹⁵)(R¹⁶), —C₃carbocycle unsubstituted or substituted with methyl or halogen,—CH(X′)—C₃ carbocycle unsubstituted or substituted with methyl orhalogen, C₃ heterocyle unsubstituted or substituted with methyl orhalogen, —CH(X′)—C₃ heterocycle unsubstituted or substituted with methylor halogen, —CH₂N₃, —CH₂CH₂N₃, and benzyloxymethyl, or R⁵ is a smallelectron withdrawing group; wherein R¹⁰ is hydrogen or C₁-C₃ alkylunsubstituted or substituted with halogen; R¹¹ is C₁-C₃ alkylunsubstituted or substituted with halogen; R¹² is hydrogen, halogen orC₁-C₃ alkyl unsubstituted or substituted with halogen; R¹³ is hydrogen,or C₁-C₃ alkyl unsubstituted or substituted with halogen; R¹⁴ ishydrogen or methyl; R¹⁵ and R¹⁶ are independently selected fromhydrogen, methyl and halogen; X is halogen; X′ is halogen or hydrogen;andadditionally, each of R¹, R², R^(2a), R³ and R^(3a) are optionallyhydrogen; optionally two R¹, R², R^(2a), R³ and R^(3a) on adjacentcarbon atoms are combined to form a double bond between said adjacentcarbon atoms; andprovided that at least one of R¹, R², R^(2a), R³, R^(3a), R⁴ and R⁵ is asmall electron withdrawing group, or R⁵ comprises a halogen, site ofunsaturation, carbocycle, heterocycle or azide, except when (i) R² andR^(2a) are both hydrogen, (ii) R³ and R^(3a) are both hydrogen, (iii) R¹is hydrogen, (iv) a double bond is present between said adjacent carbonatoms, or (v) R⁵ is benzyloxymethyl; andwherein the antibody or antibody derivative has reduced corefucosylation compared to the antibody or antibody derivative from thehost cell cultured in the absence of the fucose analog.

In some embodiments of formulae (V) and (VI), R^(2a) and R^(3a) are eachhydrogen.

In some embodiments of formulae (V) and (VI), R⁵ is selected from thegroup consisting of —CH₃, —CH₂CH₃, —CH₂C≡CH, —CH═CHCH₃, -cyclopropyl,-oxirane, -oxirane substituted with methyl, —CH₂F, —CH₂Cl, —CH₂Br,—CH₂I, —CH═C═CH₂, —CH₂N₃ and —CH₂CH₂N₃.

In some embodiments of formulae (V) and (VI), the small electronwithdrawing group is selected from fluoro, chloro, bromo, —CHF₂,—CH═C═CH₂, —C≡CH, —C≡CCH₃, —CH₂C≡CH, —CO₂H, —C(O)OC₁—C₄ alkyl,—CH(OAc)CH₃, —CN, —CH₂CN, —CH₂X (wherein X is Br, Cl or I), andmethoxiran.

In some embodiments of formulae (V) and (VI), at least two of R¹, R²,R^(2a), R³, R^(3a) and R⁴ are independently selected small electronwithdrawing groups.

In some embodiments of formulae (V) and (VI), the fucose analog isselected from compounds of Tables 1, 2 or 3.

While the present inventive methods and cell cultures can include thefucose analogs provided in formulae I, II, III, IV, V and VI above, thepresent invention further provides compounds of each of the aboveformulae that can be prepared using methodology provided herein. In someembodiments, the compounds of the invention are other than compoundsidentified in the Examples as 6, 7, 9, 10, 22, 24, 26, 54, 56-58, 61-62,65 and 66, as well as 2-fluoro-2-deoxyfucose.

Antibodies and Antibody Derivatives

Antibodies that can be produced by the instant methods can bemonoclonal, chimeric, humanized (including veneered), or humanantibodies. Suitable antibodies also include antibody fragments, such assingle chain antibodies, or the like that have a Fc region or domainhaving a complex N-glycoside-linked sugar chain (e.g., a human IgG1 Fcregion or domain). The Fc region or domain can include an Fcgammareceptor binding site. Typically, the antibodies are human or humanized.In some embodiments, the antibodies can be rodent (e.g., mouse and rat),donkey, sheep, rabbit, goat, guinea pig, camelid, horse, or chicken.

The antibodies can be mono-specific, bi-specific, tri-specific, or ofgreater multi-specificity. Multi-specific antibodies maybe specific fordifferent epitopes of different target antigens or may be specific fordifferent epitopes on the same target antigen. (See, e.g., WO 93/17715;WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al., 1991, J. Immunol.147:60-69; U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920;and U.S. Pat. No. 5,601,819; Kostelny et al., 1992, J. Immunol.148:1547-1553.)

The antibodies can also be described in terms of their binding affinityto a target antigen of 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M,5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M,10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵M.

In some embodiments, the antibody is a chimeric antibody. A chimericantibody is a molecule in which different portions of the antibody arederived from different animal species, such as antibodies having avariable region derived from a murine monoclonal antibody and a humanimmunoglobulin constant region. Methods for producing chimericantibodies are known in the art. (See, e.g., Morrison, Science, 1985,229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J.Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and4,816,397.)

In some embodiments, the antibody can be a humanized antibody, includinga veneered antibody. Humanized antibodies are antibody molecules thatbind the desired antigen and have one or more complementaritydetermining regions (CDRs) from a non-human species, and framework andconstant regions from a human immunoglobulin molecule. Often, frameworkresidues in the human framework regions will be substituted with thecorresponding residue from the CDR donor antibody to alter, orpreferably improve, antigen binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,Queen et al., U.S. Pat. No. 5,585,089; Riecbmann et al., 1988, Nature332:323.) Antibodies can be humanized using a variety of techniquesknown in the art such as CDR-grafting (EP 0 239 400; WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 0 592 106; EP 0 519 596; Padlan, 1991, Molecular Immunology,28(4/5):489-498; Studnicka et al., 1994, Protein Engineering7(6):805-814; Roguska et al., 1994, Proc. Natl. Acad. Sci. USA91:969-973), and chain shuffling (U.S. Pat. No. 5,565,332) (all of thesereferences are incorporated by reference herein).

The antibody can also be a human antibody. Human antibodies can be madeby a variety of methods known in the art such as phage display methodsusing antibody libraries derived from human immunoglobulin sequences.See e.g., U.S. Pat. Nos. 4,444,887 and 4,716,111; WO 98/46645, WO98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO91/10741. In addition, a human antibody recognizing a selected epitopecan be generated using a technique referred to as “guided selection,” inwhich a selected non-human monoclonal antibody, e.g., a mouse antibody,is used to guide the selection of a completely human antibodyrecognizing the same epitope (see, e.g., Jespers et al., 1994,Biotechnology 12:899-903). Human antibodies can also be produced usingtransgenic mice that express human immunoglobulin genes. Monoclonalantibodies directed against the antigen can be obtained from theimmunized, transgenic mice using conventional hybridoma technology. Foran overview of the technology for producing human antibodies, seeLonberg and Huszar, 1995, Int. Rev. Immunol. 13:65-93. For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Patent No. 0 598, 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; and 5,939,598.

Examples of antibodies include HERCEPTIN® (trastuzumab; Genentech),RITUXAN® (rituximab; Genentech), lintuzumab (Seattle Genetics, Inc.),Palivizumab (Medimmune), Alemtuzumab (BTG) and Epratuzumab(Immunomedics).

In exemplary embodiments, an antibody or antibody derivativespecifically binds to CD19, CD20, CD21, CD22, CD30, CD33, CD38, CD40,CD70, CD133, CD138, or CD276. In other embodiments, the antibody orantibody derivative specifically binds to BMPR1B, LAT1 (SLC7A5), STEAP1,MUC16, megakaryocyte potentiating factor (MPF), Napi3b, Sema 5b, PSCAh1g, ETBR (Endothelin type B receptor), STEAP2, TrpM4, CRIPTO, CD21,CD79a, CD79b, FcRH2, HER2, HER3, HER4, NCA, MDP, IL20Rα, Brevican,Ephb2R, ASLG659, PSCA, PSMA, GEDA, BAFF-R, CXCRS, HLA-DOB, P2X5, CD72,LY64, FCRH1, or IRTA2.

Antibodies can be assayed for specific binding to a target antigen byconventional methods, such as for example, competitive andnon-competitive immunoassay systems using techniques such as Westernblots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay),“sandwich” immunoassays, immunoprecipitation assays, immunoradiometricassays, fluorescent immunoassays, and protein A immunoassays. (See,e.g., Ausubel et al., eds., Short Protocols in Molecular Biology (JohnWiley & Sons, Inc., New York, 4th ed. 1999); Harlow & Lane, UsingAntibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1999.)

Further, the binding affinity of an antibody to a target antigen and theoff-rate of an antibody-antigen interaction can be determined by surfaceplasmon resonance, competition FACS using labeled antibodies or othercompetitive binding assays. One example of a competitive binding assayis a radioimmunoassay comprising the incubation of labeled antigen(e.g., ³H or ¹²⁵I) with the antibody of interest in the presence ofincreasing amounts of unlabeled antibody, and the detection of theantibody bound to the labeled antigen. The affinity of the antibody andthe binding off-rates can then be determined from the data by Scatchardplot analysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated with theantibody of interest conjugated to a labeled compound (e.g., ³H or ¹²⁵I)in the presence of increasing amounts of an unlabeled second antibody.Alternatively, the binding affinity of an antibody and the on- andoff-rates of an antibody-antigen interaction can be determined bysurface plasmon resonance.

Antibodies can be made from antigen-containing fragments of the targetantigen by standard procedures according to the type of antibody (see,e.g., Kohler, et al., Nature, 256:495, (1975); Harlow & Lane,Antibodies, A Laboratory Manual (C.S.H.P., NY, 1988); Queen et al.,Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989) and WO 90/07861; Doweret al., WO 91/17271 and McCafferty et al., WO 92/01047 (each of which isincorporated by reference for all purposes). As an example, monoclonalantibodies can be prepared using a wide variety of techniques including,e.g., the use of hybridoma, recombinant, and phage display technologies,or a combination thereof. Hybridoma techniques are generally discussedin, e.g., Harlow et al., supra, and Hammerling, et al., In MonoclonalAntibodies and T-Cell Hybridomas, pp. 563-681 (Elsevier, N.Y., 1981).Examples of phage display methods that can be used to make antibodiesinclude, e.g., those disclosed in Briinnan et al., 1995, J. Immunol.Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177-186;Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic et al.,1997, Gene 187:9-18; Burton et al., 1994, Advances in Immunology57:191-280; PCT Application No. PCT/GB91/01 134; PCT Publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108 (thedisclosures of which are incorporated by reference herein).

Examples of techniques that can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., 1991, Methods in Enzymology 203:46-88; Shu etal., 1993, Proc. Natl. Acad. Sci. USA 90:7995-7999; and Skerra et al.,1988, Science 240:1038-1040.

Examples of antibody derivatives include binding domain-Ig fusions,wherein the binding domain may be, for example, a ligand, anextracellular domain of a receptor, a peptide, a non-naturally occurringpeptide or the like. Exemplary fusions with immunoglobulin or Fc regionsinclude: etanercept which is a fusion protein of sTNFRII with the Fcregion (U.S. Pat. No. 5,605,690), alefacept which is a fusion protein ofLFA-3 expressed on antigen presenting cells with the Fc region (U.S.Pat. No. 5,914,111), a fusion protein of Cytotoxic TLymphocyte-associated antigen-4 (CTLA-4) with the Fc region (J. Exp.Med. 181:1869 (1995)), a fusion protein of interleukin 15 with the Fcregion (J Immunol. 160:5742 (1998)), a fusion protein of factor VII withthe Fc region (Proc. Natl. Acad. Sci. USA 98:12180 (2001)), a fusionprotein of interleukin 10 with the Fc region (J Immunol. 154:5590(1995)), a fusion protein of interleukin 2 with the Fc region (J.Immunol. 146:915 (1991)), a fusion protein of CD40 with the Fc region(Surgery 132:149 (2002)), a fusion protein of Flt-3 (fms-like tyrosinekinase) with the antibody Fc region (Acta. Haemato. 95:218 (1996)), afusion protein of OX40 with the antibody Fc region (J. Leu. Biol. 72:522(2002)), and fusion proteins with other CD molecules (e.g., CD2, CD30(TNFRSF8), CD95 (Fas), CD106 (VCAM-I), CD137), adhesion molecules (e.g.,ALCAM (activated leukocyte cell adhesion molecule), cadherins, ICAM(intercellular adhesion molecule)-1, ICAM-2, ICAM-3) cytokine receptors(e.g., interleukin-4R, interleukin-5R, interleukin-6R, interleukin-9R,interleukin-10R, interleukin-12R, interleukin-13Ralpha1,interleukin-13Ralpha2, interleukin-15R, interleukin-21Ralpha),chemokines, cell death-inducing signal molecules (e.g., B7-H1, DR6(Death receptor 6), PD-1 (Programmed death-1), TRAIL R1), costimulatingmolecules (e.g., B7-1, B7-2, B7-H2, ICOS (inducible co-stimulator)),growth factors (e.g., ErbB2, ErbB3, ErbB4, HGFR),differentiation-inducing factors (e.g., B7-H3), activating factors(e.g., NKG2D), signal transfer molecules (e.g., gp130), BCMA, and TACI.

Methods of Making Non-Core Fucosylated Antibodies and AntibodyDerivatives

Antibodies and derivatives thereof that are useful in the presentmethods can be produced by recombinant expression techniques, fromhybridomas, from myelomas or from other antibody expressing mammaliancells. Recombinant expression of an antibody or derivative thereof thatbinds to a target antigen typically involves construction of anexpression vector containing a nucleic acid that encodes the antibody orderivative thereof. Once a nucleic acid encoding such a protein has beenobtained, the vector for the production of the protein molecule may beproduced by recombinant DNA technology using techniques well known inthe art. Standard techniques such as those described in Sambrook andRussell, Molecular Cloning: A Laboratory Manual (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 3rd ed., 2001); Sambrook etal., Molecular Cloning: A Laboratory Manual (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 2nd ed., 1989); Ausubel etal., Short Protocols in Molecular Biology (John Wiley & Sons, New York,4th ed., 1999); and Glick & Pastemak, Molecular Biotechnology:Principles and Applications of Recombinant DNA (ASM Press, Washington,D.C., 2nd ed., 1998) can be used for recombinant nucleic acid methods,nucleic acid synthesis, cell culture, transgene incorporation, andrecombinant protein expression.

For example, for recombinant expression of antibody, an expressionvector may encode a heavy or light chain thereof, or a heavy or lightchain variable domain, operably linked to a promoter. An expressionvector may include, e.g., the nucleotide sequence encoding the constantregion of the antibody molecule (see, e.g., WO 86/05807; WO 89/01036;and U.S. Pat. No. 5,122,464), and the variable domain of the antibodymay be cloned into such a vector for expression of the entire heavy orlight chain. The expression vector is transferred to a host cell bytechniques known in the art, and the transfected cells are then culturedby techniques known in the art in the presence of a fucose analog toproduce the antibody. Typically, for the expression of double-chainedantibodies, vectors encoding both the heavy and light chains can beco-expressed in the host cell for expression of the entireimmunoglobulin molecule.

A variety of mammalian cells and cell lines can be utilized to expressan antibody or derivative thereof. For example, mammalian cells such asChinese hamster ovary cells (CHO) (e.g., DG44, Dxb11, CHO-K, CHO-K1 andCHO-S) can be used. In some embodiments, human cell lines are used.Suitable myeloma cell lines include SP2/0 and IR983F and human myelomacell lines such as Namalwa. Other suitable cells include human embryonickidney cells (e.g., HEK293), monkey kidney cells (e.g., COS), humanepithelial cells (e.g, HeLa), PERC6, Wil-2, Jurkat, Vero, Molt-4, BHK,and K6H6. Other suitable host cells include YB2/0 cells. In otherembodiments, the host cells are not YB2/0 cells.

In some embodiments, the host cells are from a hybridoma. In someembodiments, the host cells are not a hybridoma produced by a fusiongenerated with NS0 myeloma cells. In other embodiments, the host cellsare not from a hybridoma.

In some embodiments, the host cells do not contain a fucose transportergene knockout. In some embodiments, the host cells do not contain afucosyltransferase (e.g, FUT8) gene knockout. In some embodiments, thehost cells do not contain a knock-in of a GnTIII encoding nucleic acid.In some embodiments, the host cells do not contain a knock-in of a golgialpha mannosidase II encoding nucleic acid.

A variety of mammalian host-expression vector systems can be utilized toexpress an antibody or derivative thereof. For example, mammalian cellssuch as Chinese hamster ovary cells (CHO) (e.g., DG44, Dxb11, CHO-K1 andCHO-S) in conjunction with a vector such as the major intermediate earlygene promoter element from human cytomegalovirus or the Chinese hamsterovary EF-1α promoter, is an effective expression system for theproduction of antibodies and derivatives thereof (see, e.g., Foecking etal., 1986, Gene 45:101; Cockett et al., 1990, Bio/Technology 8:2;Allison, U.S. Pat. No. 5,888,809).

The cell lines are cultured in the appropriate culture medium. Suitableculture media include those containing, for example, salts, carbonsource (e.g., sugars), nitrogen source, amino acids, trace elements,antibiotics, selection agents, and the like, as required for growth. Forexample, commercially available media such as Ham's F10 (Sigma), MinimalEssential Medium (MEM, Sigma), RPMI-1640 (Sigma), Dulbecco's ModifiedEagle's Medium ((DMEM, Sigma), PowerCHO™ cell culture media (Lonza GroupLtd.) Hybridoma Serum-Free Medium (HSFM) (GIBCO) are suitable forculturing the host cells. Any of these media may be supplemented asnecessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleotides (such as adenosine and thymidine), antibiotics (such asGENTAMYCIN™), trace elements (defined as inorganic compounds usuallypresent at final concentrations in the micromolar range), and glucose oran equivalent energy source. Any other necessary supplements may also beincluded at appropriate concentrations that would be known to thoseskilled in the art. The culture conditions, such as temperature, pH, andthe like, can be those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisan.

The culture media preferably is not supplemented with fucose. In someembodiments, the culture media is a serum-free media. In someembodiments, the culture media is an animal-derived protein free (i.e.,animal protein free) media.

An effective amount of a fucose analog is added to the culture media. Inthis context, an “effective amount” refers to an amount of the analogthat is sufficient to decrease fucose incorporation into a complexN-glycoside-linked sugar chain of an antibody or antibody derivative byat least 10%, at least 20%, at least 30%, at least 40% or at least 50%.In some embodiments, the effective amount of the fucose analog issufficient to reduce fucose incorporation into a complexN-glycoside-linked sugar chain of an antibody or antibody derivative byat least 60%, at least 70%, at least 80% or at least 90%.

The cells expressing the antibody or antibody derivative can be culturedby growing the host cell in any suitable volume of culture mediasupplemented with the fucose analog. The cells may be cultured in anysuitable culture system and according to any method known in the art,including T-flasks, spinner and shaker flasks, WaveBag® bags, rollerbottles, bioreactors and stirred-tank bioreactors. Anchorage-dependentcells can also be cultivated on microcarrier, e.g., polymeric spheres,that are maintained in suspension in stirred-tank bioreactors.Alternatively, cells can be grown in single-cell suspension. Culturemedium may be added in a batch process, e.g., where culture medium isadded once to the cells in a single batch, or in a fed batch process inwhich small batches of culture medium are periodically added. Medium canbe harvested at the end of culture or several times during culture.Continuously perfused production processes are also known in the art,and involve continuous feeding of fresh medium into the culture, whilethe same volume is continuously withdrawn from the reactor. Perfusedcultures generally achieve higher cell densities than batch cultures andcan be maintained for weeks or months with repeated harvests.

For cells grown in batch culture, the volume of culture medium istypically at least 750 mL, 1 liter, 2 liters, 3 liters, 4 liters, 5liters, 10 liters, 15 liters, 20 liters or more. For industrialapplications, the volume of the culture medium can be at least 100liters, at least 200 liters, at least 250 liters, at least 500 liters,at least 750 liters, at least 1000 liters, at least 2000 liters, atleast 5000 liters or at least 10,000 liters. The fucose analog may beadded to the seed train, to the initial batch culture medium, after arapid growth phase, or continuously with culture medium (e.g., duringcontinuous feeding). For example, the fucose analog may be added to anearly seed train or feedstock at a 10× or 100× concentration, such thatsubsequent additions of culture media change the concentration of fucoseanalog to a level that is still effective in achieving non-corefucosylation of the antibody or antibody derivative. Alternatively, thefucose analog is added directly to the culture media, obviating the needfor dilution. In any case, the fucose analog is typically addedrelatively early in the cell culturing process and an effectiveconcentration is maintained throughout the culturing process in order tooptimize production of the desired antibody or antibody derivative.

In some embodiments, antibodies or antibody derivatives produced by theinstant methods comprise at least 10%, at least 20%, at least 30%, atleast 40% or at least 50% non-core fucosylated protein (e.g., lackingcore fucosylation), as compared with antibodies or antibody derivativesproduced from the host cells cultured in the absence of a fucose analog.In some embodiments, antibodies or antibody derivatives produced by theinstant methods comprise at least 60%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90% or at least 95% non-corefucosylated antibody or antibody derivative, as compared with antibodyor derivative produced from the host cells cultured in the absence of afucose analog. In some embodiments, a composition of antibodies orantibody derivatives produced by the instant methods comprises less than100% non-core fucosylated antibodies and/or antibody derivatives.

The amount of the fucose analog (of any of Formulae I, II, III, IV, Vand VI) that is effective can be determined by standard cell culturemethodologies. For example, cell culture assays may be employed to helpidentify optimal dosing ranges. The precise amount to be employed alsodepends on the time of administration, the host cell line, the celldensity and the like. Effective doses may be extrapolated fromdose-response curves derived from in vitro model test systems.

In some embodiments, the fucose analog is present in the culture mediumat a concentration of 10 nM to 50 mM. In some embodiments, the fucoseanalog is present in the culture medium at a concentration of 10 nM to10 mM. In some embodiments, the fucose analog is present in the culturemedium at a concentration of 100 nM to 5 mM. In some embodiments, thefucose analog is present in the culture medium at a concentration of 100nM to 3 mM. In some embodiments, the fucose analog is present in theculture medium at a concentration of 100 nM to 2 mM. In someembodiments, the fucose analog is present in the culture medium at aconcentration of 100 nM to 1 mM. In some embodiments, the fucose analogis present in the culture medium at a concentration of 1 μM to 1 mM. Insome embodiments, the fucose analog is present in the culture medium ata concentration of 10 nM to 1 mM. In some embodiments, the fucose analogis present in the culture medium at a concentration of 10 nM to 500 μM.In some embodiments, the fucose analog is present in the culture mediumat a concentration of 1 μM to 500 μM. In some embodiments, the fucoseanalog is present in the culture medium at a concentration of 1 μM to250 μM. In some embodiments, the fucose analog is present in the culturemedium at a concentration of 10 μM to 100 μM. In some embodiments, thefucose analog is soluble in the culture medium (at the appropriatetemperature for host cell maintenance/growth) at a concentration of atleast 10 nM. In some embodiments, the fucose analog is soluble in theculture medium (at the appropriate temperature for host cellmaintenance/growth) at a concentration of at least 100 nM.

The content (e.g., the ratio) of sugar chains in which fucose is notbound to N-acetylglucosamine in the reducing end of the sugar chainversus sugar chains in which fucose is bound to N-acetylglucosamine inthe reducing end of the sugar chain can be determined, for example, asdescribed in the Examples. Other methods include hydrazinolysis orenzyme digestion (see, e.g., Biochemical Experimentation Methods 23:Method for Studying Glycoprotein Sugar Chain (Japan Scientific SocietiesPress), edited by Reiko Takahashi (1989)), fluorescence labeling orradioisotope labeling of the released sugar chain and then separatingthe labeled sugar chain by chromatography. Also, the compositions of thereleased sugar chains can be determined by analyzing the chains by theHPAEC-PAD method (see, e.g., J. Liq Chromatogr. 6:1557 (1983)). (Seegenerally U.S. Patent Application Publication No. 2004-0110282.)

In some embodiments, the antibodies or antibody derivatives produce bythe instant methods have higher effector function (e.g., ADCC activity)than the antibodies or antibody derivatives produced in the absence of afucose analog. The effector function activity may be modulated byaltering the concentration of the fucose analog in the culture mediumand/or the duration of exposure to the fucose analog. ADCC activity maybe measured using assays known in the art and in exemplary embodimentsincreases by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,15-fold or 20-fold, as compared to the core fucosylated parent antibody.The cytotoxic activity against an antigen-positive cultured cell linecan be evaluated by measuring effector function (e.g., as described inCancer Immunol. Immunother. 36:373 (1993)).

Antibodies and antibody derivative can be purified using, for example,hydroxylapatite chromatography, gel electrophoresis, dialysis, andaffinity chromatography, with affinity chromatography being a preferredpurification technique. The suitability of protein A as an affinityligand depends on the species and isotype of any immunoglobulin Fcdomain that is present in the antibody or antibody derivative. Protein Acan be used to purify antibodies or antibody derivatives that are basedon human IgG1, 2, or 4 heavy chains.

Protein G can be used for mouse isotypes and for some human antibodiesand antibody derivatives. The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodyor antibody derivative comprises a C_(H)3 domain, the Bakerbond ABX™resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.Other techniques for protein purification such as fractionation on anion-exchange column (cationic or anionic exchange), ethanolprecipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody or antibody derivative to berecovered.

Following any purification step(s), the mixture comprising the antibodyor antibody derivative of interest and contaminants may be subjected tolow pH hydrophobic interaction chromatography (e.g., using an elutionbuffer at a pH between about 2.5-4.5, preferably performed at low saltconcentrations (e. g., from about 0-0.25M salt)).

Uses of the Antibodies and Antibody Derivatives

Antibodies and antibody derivatives prepared according to the presentmethods can be used for a variety of therapeutic and non-therapeuticapplications. For example, the antibodies can be used as therapeuticantibodies. Antibody derivatives (e.g., a receptor-Fc fusion) can beused as a therapeutic molecule. In some embodiments, the antibody orantibody derivative is not conjugated to another molecule. In someembodiments, the antibody is conjugated to a suitable drug (e.g., anantibody drug conjugate) or other active agent. The antibodies andantibody derivatives can also be used for non-therapeutic purposes, suchas diagnostic assays, prognostic assays, release assays and the like.

Pharmaceutical Compositions.

Antibodies and antibody derivatives prepared according to the presentmethods can be formulated for therapeutic and non-therapeuticapplications. The antibodies and derivatives can be formulated aspharmaceutical compositions comprising a therapeutically orprophylactically effective amount of the antibody or derivative and oneor more pharmaceutically compatible (acceptable) ingredients. Forexample, a pharmaceutical or non-pharmaceutical composition typicallyincludes one or more carriers (e.g., sterile liquids, such as water andoils, including those of petroleum, animal, vegetable or syntheticorigin, such as peanut oil, soybean oil, mineral oil, sesame oil and thelike). Water is a more typical carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable excipients include, forexample, amino acids, starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol, and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.Such compositions will typically contain a therapeutically effectiveamount of the protein, typically in purified form, together with asuitable amount of carrier so as to provide the form for properadministration to the patient. The formulations correspond to the modeof administration.

Typically, compositions for intravenous administration are solutions insterile isotonic aqueous buffer. When necessary, the pharmaceutical canalso include a solubilizing agent and a local anesthetic such aslignocaine to ease pain at the site of the injection. Generally, theingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. When thepharmaceutical is to be administered by infusion, it can be dispensedwith an infusion bottle containing sterile pharmaceutical grade water orsaline. When the pharmaceutical is administered by injection, an ampouleof sterile water for injection or saline can be provided so that theingredients can be mixed prior to administration.

The invention is further described in the following examples, which arenot intended to limit the scope of the invention.

EXAMPLES Example 1: Synthesis of Alkynyl Fucose Peracetate and GeneralProcedure for Synthesis of Fucose Analogs

The preparation of alkynyl fucose peracetate (also referred to asperacetyl alkynyl fucose and alkynyl peracetyl fucose) (Compound 7) wasdescribed by Sawa et al., 2006, Proc. Natl. Acad. Sci. USA103:12371-12376 and Hsu et al., 2007, Proc. Natl. Acad. Sci. USA104:2614-2619, with the following modification. A Corey-Fuchshomologation sequence was employed to install the alkynyl group, asdescribed by Pelphrey et al., 2007, J. Med. Chem. 50:940-950.

General methods for other fucose analogs: Common reagents and solventswere purchased from either Fisher or Sigma-Aldrich except as follows:L-galactono-1,4-lactone was purchased from Carbosynth Limited. ¹H-NMRspectra were recorded on a Varian Mercury spectrometer at 400 MHz. LC/MSdata were obtained on a Waters Micromass instrument using an HP 1100HPLC with a PDA detector. The column used was a Phenomenex SynergiMaxRP-C12 column (2 mm×150 mm) eluting with a MeCN-water gradientcontaining 0.05% formic acid. Flash column chromatography (FCC) wasperformed using 230-400 mesh ASTM silica gel from EM Science or using aChromatotron. Analtech silica gel GHLF plates were used for thinlayerchromatography and TLC plates were stained with vanillin or iodine. HPLCwas performed using a Waters Alliance system with a PDA detector.

Example 2: Antibody Expression in the Presence of Alkynyl FucosePeracetate

To determine the effect of alkynyl fucose peracetate on glycosylation ofantibodies, a CHO DG44 cell line expressing a humanized IgG1 anti-CD70monoclonal antibody, h1F6 (see International Patent Publication WO06/113909) was cultured at 3.0×10⁵ cells per mL in 30 mLs of CHO culturemedia at 37°, 5% CO₂, by shaking at 100 RPM in 125 mL shake flasks. TheCHO culture media was supplemented with insulin like growth factor(IGF), penicillin, streptomycin and either 50 or 100 μM alkynyl fucoseperacetate (prepared as described in Example 1). Cultures were fed onday 3 with 2% volume of feed media containing 2.5 or 5 mM alkynyl fucoseperacetate for the 50 and 100 μM alkynyl fucose peracetate cultures,respectively. On day four, each culture was split 1:4 into fresh culturemedia. Cultures were fed with a 6% volume of feed media containing 833μM or 1.66 mM alkynyl fucose peracetate on days 5, 7, 9 and 10.Conditioned media was collected on day 13 by passing the media through a0.2 μm filter.

Antibody purification was performed by applying the conditioned media toa protein A column pre-equilibrated with 1× phosphate buffered saline(PBS), pH 7.4. After washing the column with 20 column volumes of 1×PBS,antibodies were eluted with 5 column volumes of Immunopure IgG elutionbuffer (Pierce Biotechnology, Rockford, Ill.). A 10% volume of 1M trispH 8.0 was added to eluted fraction. The sample was dialyzed overnightinto 1×PBS.

Example 3: LC-MS (Q-Tof) Analysis of Antibodies Produced by Expressionin the Presence of Alkynyl Fucose Peracetate

To identify the glycosylation pattern present on purified h1F6antibodies from Example 2, antibody interchain disulfide bonds werereduced by adding 10 μL of 100 mM DTT to 90 μL of 1 mg/mL antibody inPBS and incubation at 37° C. for 15 min. This solution (20 μL) wasinjected onto a PLRP-S HPLC column (Polymer Laboratories; Amherst,Mass.) running the following gradient: solvent A, 0.05% TFA in water;solvent B, 0.035% TFA in acetonitrile; a linear gradient of 70 to 50% Afrom 0 to 12.5 min. The HPLC effluent was analyzed with an electrosprayionization Q-Tof mass spectrometer (Waters, Milford, Mass.) with a conevoltage of 35 V collecting from m/z 500 to 4000. Data for the heavychain were deconvoluted using the MaxEntl function in MassLynx 4.0.

Surprisingly, the heavy chains of antibodies from cells grown in thepresence of alkynyl fucose peracetate showed a decrease by about 146 Da,as compared to control antibodies (i.e., heavy chains of antibodies fromcells grown in the absence of alkynyl fucose peracetate). Thisobservation suggested that addition of alkynyl fucose peracetate to theculture did not grossly alter the glycosylation pattern of theantibodies. Instead, addition of alkynyl fucose peracetate caused aminor but detectable change in glycosylation. The change in mass isconsistent with the absence of fucose in the antibodies.

Example 4: Capillary Electrophoresis of Oligosaccharides

To further characterize the glycans on the h1F6 antibodies from Example3, capillary electrophoresis was performed. Samples of the antibodieswere buffer-exchanged into water. 300 μg of each sample was treated withPNGaseF overnight at 37° C. to release oligosaccharides. The proteincomponent of the sample was removed by addition of cold methanol (−20°C.) and centrifuged for 10 minutes at 14,000 rpm. The supernatant wasdried and oligosaccharides were labeled using APTS(8-aminopyrene-1,3,6-trisulfonic acid, trisodium salt) in 1M sodiumcyanoborohydride/THF at 22° C. overnight. Labeled oligosaccharides werediluted with water and analyzed by capillary electrophoresis using aBeckman Coulter PA-800 in a N—CHO coated capillary (Beckman Coulter).For FIG. 1A, the samples were run in N-linked gel buffer (BeckmanCoulter, Fullerton, Calif., USA). For FIGS. 1B and 1C, the samples wererun in 40 mM EACA, 0.2% HPMC at pH 4.5. Samples were injected for 8seconds at 0.5 psi and separated at 30 kV for 15 minutes. Labeledoligosaccharides were detected using laser induced fluorescence (LFI)with an excitation wavelength of 488λ. Emission fluorescence wasdetected at 520λ.

Samples of the antibodies were also treated with β-galactosidase toremove galactose. The antibody samples were buffer exchanged into water.300 μg of each sample was treated with PNGaseF overnight at 37° C. torelease oligosaccharides. The protein component of the sample wasremoved by addition of cold methanol (−20° C.) and centrifugation for 10minutes at 14,000 rpm. The supernatants were dried, resuspended in waterand treated with β-galactosidase. Oligosaccharides were dried and thenlabeled using APTS in 1M sodium cyanoborohydride/THF at 22° C.overnight. Labeled oligosaccharides were diluted with water and analyzedby capillary electrophoresis using a Beckman Coulter PA-800, in a N—CHOcoated capillary (Beckman Coulter) running in 40 mM EACA, 0.2% HPMC atpH 4.5. Samples were injected for 8 seconds at 0.5 psi and separated at30 kV for 15 minutes. Labeled oligosaccharides were detected using laserinduced fluorescence (LFI) with an excitation wavelength of 488λ.Emission fluorescence was detected at 520λ.

An analysis of the data from the capillary electrophoresis is shown inFIG. 1. Referring to FIG. 1A, the electropherogram of glycans from thecontrol h1F6 antibody are shown. FIG. 1B shows an electropherogram ofglycans from the h1F6 antibody produced from a host cell grown in thepresence of alkynyl fucose peracetate. A comparison of FIGS. 1A and 1Breveals increased amounts of non-core fucosylated G0-F (and acorresponding decrease in core fucosylated G0 and G1 levels). Becausethe non-core fucosylated G1 peak co-migrated with the core fucosylatedG0, it was difficult to determine the relative distribution of thedifferent glycans. To de-convolute the data, separate antibody sampleswere treated with β-galactosidase. Referring to FIG. 1C, removing thegalactose effectively collapsed the electropherogram to two peaks, G0and G0-F (lacking fucose). In this β-galactosidase treated sample,approximately 85% of the oligosaccharide is non-core fucosylated and 6%is core fucosylated. The remainder consists of minor species.

Example 5: Antibody Dependent Cellular Cytotoxicity (ADCC) Assay

To confirm that some of the h1F6 antibody produced in Example 2 was notcore fucosylated (as compared to the parent antibody), the activity ofthe antibody was determined by an ADCC assay. The ADCC activity assaywas a standard ⁵¹Cr release assay, as described previously (seeMcEarchem et al., Blood 109:1185 (2007)). Briefly, 786-O cell linetarget tumor cells were labeled with 100 μCi Na[⁵¹Cr]O₄ and then washed.Effector (NK) cells were prepared from non-adherent peripheral bloodmononuclear cells (PBMCs) obtained from normal FcγRIIIA 158V donors(Lifeblood, Memphis, Tenn.). The cell fraction was enriched for CD16⁺ NKcells following centrifugation over a Ficoll-Paque density gradient byremoval of T, B, and monocyte subsets and negative depletion of CD4,CD8, CD20, and CD14+ cells using immunomagnetic beads (EasySep, StemCellTechnologies, Vancouver, BC, Canada). Na₂[⁵¹Cr]O₄-labeled 786-O targettumor cells were mixed with mAb and the CD16+ effector cells at aneffector:target cell ratio of 10:1.

After a 4 h incubation at 37° C., the radioactivity (⁵¹Cr) released intothe culture supernatant was measured and the percent specific cell lysiscalculated as (test sample cpm−spontaneous cpm)/(total cpm−spontaneouscpm)×100. Spontaneous and total cpm were determined from thesupernatants of target cells incubated in medium alone and from targetcells lysed with 1% triton-X100, respectively.

Referring to FIG. 2, in the ADCC assay using PBMC as a source of naturalkiller (NK) cells (having the 158V phenotype), control anti-CD70 mAb(shaded circles) lysed CD70+ target cells in a dose dependent fashion,while no lysis was observed with nonbinding control human IgG (shadeddiamonds). In contrast, anti-CD70 antibody isolated from host cellsgrown in the presence of alkynyl fucose peracetate (“AlkF”) has enhancedADCC activity (open circles and triangles). The half maximal lysis(EC₅₀) of control anti-CD70 antibody was about 9 ng/mL while the EC₅₀concentrations of mAb produced in the presence of 50 μM and 100 μM AlkFwere 0.5 and 0.3 ng/mL, respectively. The latter antibodies also gaverise to higher maximal specific lysis (53.3±3.8 and 54.8±4.7 percent)compared to that achieved with control anti-CD70 mAb (42.5±5.8 percent).

Example 6: FcγR Binding Assays

Fcγ receptor binding assays were performed to compare the binding ofcontrol CD70 antibody with the non-core fucosylated antibodies ofExample 2. Briefly, stable CHO DG-44 cell lines expressing humanFcγRIIIA V158 or murine FcγRIV were combined with 50 nmol/L or 200nmol/L Alexa Fluor 488 labeled anti-CD70 IgG1, respectively, in thepresence of serial dilutions of each of the following anti-CD70antibodies in PBS, 0.1% BSA (w/v) buffer: control h1F6 antibody, andh1F6 antibody from host cells cultured with alkynyl fucose peracetate.The mixtures were incubated for 60 minutes on ice in the dark. Labeledcells were detected using an LSRII FACS analyzer and data were analyzedby a nonlinear least squares fit to a 4-parameter logistic equationusing Prism v5.01 to estimate EC₅₀ values.

Non-core fucosylated anti-CD70 antibodies (triangles) competed forbinding to huFcγ receptors (FIG. 3A) and muFcγ receptors (FIG. 3B) withfluorescently-labeled anti-CD70 parent antibody (squares). The non-corefucosylated anti-CD70 out-competed the parent (control) anti-CD70antibody for binding to the murine receptor, muFcγRIV, with EC₅₀ valuesof 20.8 nM and 368.9 nM, respectively (an 18 fold difference). Non-corefucosylated anti-CD70 also out-competed the parent antibody in bindingto the human receptor, huFcγRIIIA V158, with EC₅₀ values of 7.99 nM and112.9 nM, respectively (a 14-fold difference).

Example 7: Expression of Other Antibodies in the Presence of AlkynylFucose Peracetate

To confirm the effect of alkynyl fucose peracetate on glycosylation ofadditional antibodies, antibodies were expressed from the following celllines: CD70 Ab h1F6 in DG44 cells; CD19 Ab hBU12 in DG44 cells (see U.S.Provisional Application No. 61/080,169, filed Jul. 11, 2008); CD30 AbcAC10 in DG44 cells; and CD33 Ab HuM195 in SP2/0 and CHO-K1 cell (seealso U.S. Ser. No. 12/253,895, filed Oct. 17, 2008). Briefly, the celllines were initially cultured at 3.0×10⁵ cells per mL in 30 mLs of CHOselection media at 37° C., 5% CO₂ and shaking at 100 RPM. The media wassupplemented with insulin like growth factor (IGF), penicillin,streptomycin and 50 μM alkynyl fucose peracetate, as described. Thecultures were fed on day 3 with 2% volume of feed media containing 2.5mM alkynyl fucose peracetate. On day four, the cultures were split 1:4into fresh culture media. Cultures were fed with a 6% volume of feedmedia containing 833 μM alkynyl fucose peracetate on days 5, 7, 9 and10. Conditioned media was collected on day 13 by passing the culturethrough a 0.2 μm filter.

Antibody purification was performed by applying the conditioned media toa protein A column—pre-equilibrated with 1× phosphate buffered saline(PBS), pH 7.4. Antibodies were eluted with 5 column volumes ofImmunopure IgG elution buffer (Pierce Biotechnology, Rockford, Ill.). A10% volume of 1M tris pH 8.0 was added to the eluted fraction. Thesample was dialyzed overnight into 1×PBS.

Qtof analysis of the antibodies revealed similar results to those ofExample 3. Relative to heavy chains of antibodies produced from hostcells grown in the absence of alkynyl fucose peracetate, heavy chains ofantibodies from cells grown in the presence of alkynyl fucose peracetatewere observed to decrease by about 146 Da, consistent with the absenceof fucose. Referring to FIG. 4, for the G0 peak (no galactose) for eachantibody, the observed shift in mass between heavy chain from cellsgrown in the absence and presence of alkynyl fucose peracetate (upperand lower portions of each panel) was a decrease of 144 Da (anti-CD70antibody, FIG. 4A), 145 Da (anti-CD19 antibody, FIG. 4B), 146 Da(anti-CD30 antibody, FIG. 4C), and 146 Da (anti-CD33 antibody, FIG. 4D).These decreases in molecular weight are inconsistent with loss of anyother sugar found in antibody carbohydrate other than fucose: mannoseand galactose, 162 Da, N-acetylglucosamine, 203 Da, and sialic acid, 291Da.

Example 8: Effector Function Assays

The effector functions, ADCC and ACCP, of a non-core fucosylated,humanized CD19 antibody, hBU12, was determined. ADCC activity wasgenerally measured as described in Example 5 using Ramos cells. NK cellswere isolated from individuals with 158V and 158F FcγRIIIa phenotypes.

Antibody-dependent cellular phagocytosis (ADCP) was assessed using apreviously described method (see McEarchern et al., Blood 109:1185(2007)). Briefly, target Ramos cells were incubated with the fluorescentdye PKH26 (Sigma, St. Louis, Mo.) prior to addition of the antibody andprimary human macrophages. Macrophages were generated from normal humanPBMCs cultured for 10 to 14 days with 500 U/ml human G-MCSF (PeproTech,Rocky Hill, N.J.). After a 1 h incubation at 37° C., the macrophageswere labeled with a FITC-conjugated CD11b antibody (BD Pharmingen).Uptake of the target cells by the macrophages (phagocytosis) wasassessed by flow cytometry and visualized by immunofluorescence using aCarl Zeiss Axiovert 200M microscope. Specific phagocytosis wasdetermined by correcting for the hIgG1 background values.

Referring to FIGS. 5A and 5B, the non-core fucosylated CD19 antibody(closed triangles) exhibited an approximately 100-fold increase in EC₅₀in the 158 V background, with a 3.5-fold increase in maximum target celllysis, as compared with the control (core fucosylated) antibody (closedsquares). In the 158F background, the non-core fucosylated CD19 antibody(open triangles) had a 100 fold increase in ECso and a 10-fold increasein maximum target cell lysis, as compared with the control (corefucosylated) antibody. In contrast, no change in ACDP activity wasobserved between the non-core fucosylated and control antibody (data notshown).

Example 9: Expression of Antibodies from Hybridomas

Three antibody expressing hybridoma lines were tested to determine theeffect of alkynyl fucose peracetate on antibody core fucosylation fromthese cell lines. These hybridomas were: 1) a BALB/C mouse spleen celland a P2X63-AG 8.653 mouse myeloma cell fusion expressing the chimericanti-ley antigen antibody BR96; 2) a BALB/C mouse spleen cell and a NS0mouse myeloma cell fusion expressing a murine anti-Liv1 antibody; and 3)a BALB/C mouse spleen cell and a SP2/0 mouse myeloma cell fusionexpressing a murine anti-Liv-1 antibody. These hybridomas were culturedat 3.0×10⁵ cells per mL in 30 mLs of Hybridoma Serum Free Media(Invitrogen, Carlsbad Calif.) supplemented with 50 LM alkynyl fucoseperacetate at 37° C., 5% CO₂ and shaking at 100 RPM in a 125 mL shakeflask. Cultures were fed on day 3 with 2% volume of a feed media. On dayfour, the culture was split 1:4 into fresh culture media. Cultures werefed with a 6% volume of feed media on days 5, 7, 9 and 10. Conditionedmedia was collected when the viability of the culture dropped below 60%or on day 13 by passing culture through a 0.2 μm filter.

Antibody purification was performed by applying the conditioned media toa protein A column pre-equilibrated with 1× phosphate buffered saline(PBS), pH 7.4. After washing column with 20 column volumes of 1×PBS,antibodies were eluted with κ column volumes of Immunopure IgG elutionbuffer (Pierce Biotechnology, Rockford, Ill.). A 10% volume of 1M trispH 8.0 was added to eluted fraction. The sample was dialyzed overnightinto 1×PBS.

At the concentration of alkyl fucose peracetate tested, corefucosylation was inhibited in the hybridoma from the BALB/C-SP2/0fusion, but not the BALB/C/P2X63-AG 8.653 and NS0 fusions.

Example 10: Synthesis of 5-ethynylarabinose Tetraacetate (7)

1,2:3,4-Di-O-isopropylidene-α-L-galactose (2)

The compounds in Scheme 1 were generally prepared as described by Hsu etal., Proc. Natl. Acad. Sci. USA 104:2614-19 (2004). Briefly,L-galactono-1,4-lactone (1) (10 g, 56.1 mmol) in CH₃OH (60 ml) wascombined with water (250.0 ml) at 0° C. and Amberlite IR 120 (H+) resin(10 g). NaBH₄ (1.0 equiv. 2.22 g, 56 mmol) was added portion wise overthe course of 1 h (6 additions) with slow stirring. After NaBH₄ additionwas complete, the reaction mixture was slowly stirred for 1 h at 0° C.and then stirred vigorously at 0° C. for 15 min to promote thedecomposition of the remaining NaBH₄. The liquid was decanted, the resinwashed with methanol (2×25 mL) and the solution concentrated underreduced pressure and then under high vacuum overnight resulting in theformation of a glass. To the resulting solid was added acetone (220.0ml), CuSO₄ (22 g) and H₂SO₄ (2 ml) and the solution was stirredvigorously at room temperature for at least 24 h. After 24 h, inspectionby TLC (50% ethyl acetate in hexanes) showed product formation bystaining with vanillin dip stain and heat (R_(f)˜0.5). The reactionmixture was neutralized with Ca(OH)₂ or Cu(OH)₂ (˜15 g) and vacuumfiltered. The residue was purified by flash radial chromatography with agradient elution from 10% to 50% ethyl acetate in hexanes. Cleanfractions were combined to afford 3.3 g; (23%): ¹H NMR (CD₃OD, 400 MHz)δ: 5.48 (d, J=4.5 Hz, 1H), 4.62 (dd, J=7.8, 2.3 Hz, 1H), 4.24 (dd,J=4.9, 2.3 Hz), 4.27 (dd, J=8.0, 1.8 Hz), 3.85 (m, 1H), 3.64 (m, 2H),1.51 (s, 3H), 1.39 (s, 3H), 1.32 (s, 6H).

1,2:3,4-di-O-isopropylidene-α-L-galactal Pyranoside (3)

A suspension of pyridinium chlorochromate (PCC) (8.2 g, 38 mmol), sodiumacetate (6.2 g, 76 mmol) and 4-A molecular sieves (16 g) in drymethylene chloride (114 ml) was stirred for 1 h. To this mixture wasadded a solution of the alcohol (Compound 2) (3.3 g, 12.7 mmol) in drymethylene chloride (57 ml) drop-wise, and the mixture was stirred atroom temperature for 2 h. The reaction mixture was diluted withhexane/ether (1:1, 300 ml), and the solution was filtered through a bedof silica gel. The filter pad was washed with ethyl acetate (200 mL).The filtrate was concentrated under reduced pressure and high vacuumovernight to give 2.9 g (88%): ¹H NMR (C₆D₆; 400 MHz) δ 9.61 (s, 1H),5.44 (d, J=5.1 Hz, 1H), 4.27 (dd, J=2.3 Hz, 1H), 4.24 (dd, J=2.3 Hz,1H), 4.13 (d, J=2.3 Hz, 1H), 4.04 (dd, J=2.3 Hz, 1H), 1.32 (s, 3H), 1.2(s, 3H), 0.98 (s, 3H), 0.93 (s, 3H).

Dibromo-Olefin (4)

To a flame-dried 25 mL round-bottom flask was added CBr₄ (2.38 g, 7.14mmol) and methylene chloride (50 mL). The solution was cooled to 0° C.and Ph₃P (3.71 g, 14.3 mmol) was added. The mixture was stirred for 15min, and the aldehyde (Compound 3) (0.62 g, 2.38 mmol) was added as asolution in methylene chloride (5 mL). The reaction was monitored byTLC. After 5 min., the reaction was complete. The mixture wasconcentrated under reduced pressure to approximately 5 mL and this wasdirectly purified by flash column chromatography with 10% followed by25% ethyl acetate in hexanes. The product-containing fractions (stainsdark purple with vanillin stain (R_(f)=0.5 in 25% ethyl acetate inhexanes)) were combined and concentrated to give 495 mg. (51%): ¹H NMR(CDCl₃; 400 MHz) δ: 6.86 (d, J=8.2 Hz, 1H), 5.39 (d, J=4.9 Hz, 1H), 4.62(dd, J=8.0, 1.8 Hz, 1H), 4.37 (dd, J=7.8, 2.3 Hz), 4.03 (dd, J=5.1, 2.5Hz, 1H), 3.90 (dd, J=5.8, 2.0 Hz, 1H), 1.1 (s, 3H), 1.0 (s, 3H), 0.67(s, 3H), 0.63 (s, 3H).

6,7-Deoxy-1,2:3,4-di-O-isopropylidene-α-L-galacto-hept-6-ynopyranoside(5)

To the dibromo olefin (Compound 4) (500 mg, 1.2 mmol) in THF (15 mL) at−78° C. was added n-BuLi (3.0 mL of a 1.6M solution in hexanes; 4.87mmol) and the reaction was stirred for 1 h before being treated with asolution of ammonium chloride. The layers were separated and the aqueouswas extracted with hexanes (3×50 mL). The combined extracts were washedwith brine and dried over Na₂SO₄, filtered and concentrated. Theresulting residue was purified via radial chromatography to give 483 mg(79%): ¹H NMR (CDCl₃; 400 MHz) δ: 5.39 (d, J=5.0 Hz, 1H), 4.69 (t, J=2.3Hz, 1H), 4.36 (dd, J=7.6, 2.5 Hz), 4.01 (dd, J=5.0, 2.5 Hz, 1H), 3.94(dd, J=7.6, 2.0 Hz, 1H), 2.01 (d, J=2.3 Hz, 1H), 1.50 (s, 3H), 1.23 (s,3H), 1.11 (s, 3H), 0.92 (s, 3H).

5-ethynylarabinose (6)

To a flask containing the alkyne (Compound 5) (0.483 g, 1.9 mmol), TFAsolution (10 ml, 90% TFA in H₂O) was slowly added at 0° C. The reactionwas stirred on ice for 1 h and concentrated in vacuo.

5-Ethynylarabinose Tetraacetate (7)

(General Procedure) The resulting residue of 5-ethynylarabinose(Compound 6) was treated with pyridine (10 ml),N,N,dimethylaminopyridine (5.0 mg), and acetic anhydride (10 ml),stirred overnight, concentrated to a residue and diluted withdichloromethane. The mixture was aspirated onto a 4 mm radialchromatotron plate and eluted with 25% followed by 50% ethyl acetate inhexanes. The product was isolate as an inseparable mixture of pyranoseand furnanose α and β-anomers. Yield: 495 mg (76%): LRMS (ESI⁺) m/z 365(M+Na)⁺, 283 (M-OAc)⁺

Example 11. Synthesis of 6-Methyl-L-galactose Pentaacetate

1,2:3,4-di-O-isopropylidene-α-L-6-methylgalactopyranoside (8)

Referring to Scheme 2, to a flame-dried flask was added ether (2 mL) andCH₃MgBr (258 μL of a 3M solution). This was followed by the addition ofthe aldehyde (Compound 3) (100 mg) in ether (2 mL), added drop-wise. Thereaction mixture was stirred at room temperature for several hours andwas monitored by TLC. The reaction mixture was quenched with saturatedaqueous ammonium chloride and the mixture was extracted with ether (3×50mL). The combined extracts were washed with water and brine and driedover MgSO₄. Filtration and concentration gave a residue that wasanalyzed by ¹H NMR revealing a diastereomeric mixture. The residue waspurified via radial chromatography on a 1 mm plate eluting with 10% to25% ethyl acetate in hexanes (R_(f)=0.2; 25% ethyl acetate in hexanes).The yield was 59 mg (55%): ¹H NMR-major isomer (CDCl3; 400 MHz) δ: 5.61(d, 1H), 4.62 (dd, 1H), 4.38 (d, 1H), 4.73 (dd, 1H), 4.04 (m, 1H), 1.56(s, 3H), 1.50 (s, 3H), 1.37 (s, 3H), 1.28 (d, 3H).

6-Methyl-L-galactose Pentaacetate (9)

Compound 9 was prepared from Compound 8 by following the generalprocedure for acetonide hydrolysis and peracetylation in Example 10.LRMS (ESI⁺) m/z 345 (M-OAc)⁺.

Example 12: Synthesis of L-galactose Pentaacetate

L-galactose pentaacetate (10)

Compound 10 was synthesized from Compound 2 following the generalprocedure for acetonide hydrolysis and peracetylation in Example 10.(49% overall): LRMS (ESI⁺) m/z 331 (M-OAc)⁺.

Example 13: Synthesis of 5-vinylarabinose Tetraacetate

6,7-Deoxy-1,2:3,4-di-O-isopropylidene-α-L-galacto-hept-6-enopyranoside(11)

Referring to Scheme 4, to a −78° C. solution of Ph₃PCH₃Br (192 mg, 0.54mmol) in THF (2 mL) was added n-BuLi (0.34 mL of a 1.6M solution in THF;0.54 mmol). The mixture was stirred at −78° C. for 15 min, followed bythe addition of aldehyde (Compound 3) (46.3 mg, 0.18 mmol). The mixturewas allowed to warm to an ambient temperature over 1.5 h before beingdiluted with diethyl ether (25 mL) and quenched with saturated aqueousammonium chloride (25 mL). The organic layer was washed with brine,dried over magnesium sulfate, filtered and concentrated under reducedpressure. The resulting residue was purified on silica gel (10% ethylacetate in hexane; TLC R_(f)=0.56 25% ethyl acetate in hexanes) to give22.8 mg (49%): ¹H NMR (CDCl₃; 400 mHz) δ: 5.93 (ddd, 1H), 5.59 (d, 1H),5.37 (dt, 1H), 5.28 (dt, 1H), 4.62 (dd, 1H), 4.31 (m, 2H), 4.23 (dd,1H), 1.54 (s, 3H), 1.47 (s, 3H), 1.35 (s, 3H).

5-vinylarabinose Tetraacetate (12)

Compound 12 was prepared from Compound 11 following the generalprocedure for acetonide hydrolysis and peracetylation of Example 10.Yield: 62% overall.

Example 14: Synthesis of 5-(1-propynyl)-arabinose Tetraacetate

6,7-Deoxy-1,2:3,4-di-O-isopropylidene-α-L-galacto-oct-6-ynopyranoside(13)

Referring to Scheme 5, to a −78° C. solution of the dibromo-olefin(Compound 4) (52 mg, 0.126 mmol) in THF (1.6 mL) was added n-BuLi (0.3mL of a 1.6M solution in THF; 0.5 mmol) and the mixture was stirred for1 h at −78° C. A solution of methyl iodide (47 μL, 0.63 mmol) in THF(0.5 mL) was added drop-wise, and the reaction mixture was allowed towarm to an ambient temperature over several hours. The reaction mixturewas again cooled to −78° C. and saturated aqueous ammonium chloride (20mL) was added. The resulting mixture was extracted with ethyl acetate(3×20 mL) and the combined organics were washed with saturated aqueoussodium chloride, dried of magnesium sulfate, filtered and concentratedunder reduced pressure. The resulting residue was purified on silica geleluting with 10% ethyl acetate in hexanes to give 16.9 mg (50%) ofproduct as an oil.

5-(1-propynyl)-arabinose-1,2,3,4-tetraacetate (14)

Compound 14 was prepared from Compound 13 following the generalprocedure for acetonide hydrolysis and peracetylation in Example 10.Yield: 58% overall.

Example 15: Synthesis of 5-cyano-arabinopyranose-1,2,3,4-tetraacetateand 5-cyano-arabinofuranose-1,2,3,5-tetraacetate

1,2:3,4-Di-O-isopropylidene-α-L-galactodialdo-1,5-pyranose-6-oxime (15)

Compound 15 was generally prepared as described by Streicher and Wunsch,Carbohydr Res. 338(22):2375-85 (2003). Referring to Scheme 6, thealdehyde (Compound 3) (200 mg, 0.77 mmol), hydroxylamine hydrochloride(161 mg, 2.32 mmol, 3.0 eq.), and NaOAc (127 mg, 1.55 mmol, 2.0 eq.)were diluted in MeOH (10 mL, 0.1 M) followed by the addition of water (1mL, 10% v/v). The reaction stood for 20 h. The mixture was concentrateddown to the aqueous layer to which it was extracted with ether (2×). Thecombined organics were extracted with NaOH (1×), and the aqueous layeracidified to pH 4-5 with 1 M HCl. The aqueous phase was extracted withether (3×). The Et₂O layer was dried (MgSO₄) and concentrated in vacuoto provide the product as a white crystalline solid. Yield: (164 mg,78%). LRMS (ESI⁺) m/z 274.1 (M+H)⁺. ¹H-NMR (CDCl₃) δ: 1:1 mixture of(E):(Z) oxime isomers was detected. 1.33 (s, 12H), 1.46 (s, 6H), 1.54(s, 3H), 1.55 (s, 3H), 4.29 (dd, J=2.0 Hz, 8.0 Hz, 1H), 4.34-4.36 (m,2H), 4.43 (dd, J=2.0 Hz, 6.4 Hz, 1H), 4.62-4.63 (m, 3H), 5.00 (d, J=4.0Hz, 1H), 5.55 (d, J=4.8 Hz, 2H), 6.80 (d, J=4.4 Hz, 1H), 7.46 (d, J=6.0Hz, 1H).

5-cyano-fucose Diacetonide (16)

Compound 16 was generally prepared as described by Telvekar, SyntheticCommunications 34(13):2331-2336 (2004). The oxime isomers (Compound 15)(160 mg, 0.5 mmol) were dissolved in CH₂Cl₂ (2 mL, 0.4 M). To this wasadded a solution of benzotriazole (70 mg, 0.5 mmol) and thionyl chloride(43 μL, 0.5 mmol) in 0.5 mL of CH₂Cl₂. The reaction was complete by TLCanalysis in 5 min. The contents were filtered and the filtrate washedwith sat. NaHCO₃ and water. The organic phase was dried (Na₂SO₄),filtered and evaporated. The crude product was purified by FCC elutingwith 4:1 hexanes-EtOAc. Yield: 120 mg (81%). LRMS (ESI⁺) m/z 256.1(M+H)⁺. ¹H-NMR (CDCl₃) δ: 1.35 (s, 3H), 1.39 (s, 3H), 1.54 (s, 3H), 1.55(s, 3H), 4.34 (dd, J=2.0 Hz, 7.6 Hz, 1H), 4.38 (dd, J=2.8 Hz, 4.8 Hz,1H), 4.66 (m, 2H), 5.54 (d, J=5.2 Hz, 1H).

5-Cyano-arabinopyranose-1,2,3,4-tetraacetate (19) and5-cyano-arabinofuranose-1,2,3,5-tetraacetate (20)

Compounds 19 and 20 were prepared from Compound 16 following the generalprocedure for acetonide hydrolysis and peracetylation of Example 10. Theresulting pyranose and furanose forms were separable by FCC (elutiongradient—4:1 to 3:2 hexanes-EtOAc). Sequence of elution by TLC: pyranose(Rf 0.34), furanose (Rf.27) in 3:2 hexane-EtOAc. Yield: 59 mg(pyranose), 52 mg (furanose) (98% combined overall yield). LRMS (ESI⁺)m/z. 284.1 (M-OAc)⁺, 366.0 (M+Na)⁺. ¹H-NMR assignments were analogous tothe alkynyl fucose reported by Hsu et al. (Hsu, Hanson et al., 2007;supra). ¹H-NMR summary of pyranose forms (CDCl₃) δ: 5.89 (d, J=4.0 Hz,1H, β-pyr), 6.42 (d, J=2.8 Hz, 1H, α-pyr). ¹H-NMR summary of furanoseforms (CDCl₃) δ: 6.27 (s, 1H, α-fur), 6.38 (d, J=4.8 Hz, 1H, β-fur).

Example 16: Synthesis of chloro-, bromo- and iodo-fucose Tetraacetates

6-Chloro-fucose Diacetonide (21)

Referring to Scheme 7, the alcohol (Compound 2) (100 mg, 0.384 mmol),CCl₄ (1 mL, 10 mmol), and PPh₃ (300 mg, 1.15 mmol, 3 eq.) were dissolvedin CHCl₂ (2 mL). The contents were stirred for 24 h following byconcentration. The residue was purified by FCC (9:1 hexanes-EtOAc) toafford the product as a pale yellow gel. Yield: 107 mg (55%). LRMS(ESI⁺) m/z 279 (M+H)⁺. ¹H-NMR (CDCl₃) δ: 1.34 (s, 3H), 1.36 (s, 3H),1.45 (s, 3H), 1.54 (s, 3H), 3.58 (dd, J 6.8 Hz, 10.8 Hz, 1H), 3.68 (dd,J=6.8 Hz, 10.8 Hz, 1H), 4.65 (dd, J=2.4 Hz, 7.6 Hz, 1H), 5.54 (d, J=5.2Hz).

6-Chloro-fucose Tetraacetate (22)

Compound 22 was prepared from Compound 21 following the generalprocedure for acetonide hydrolysis and peracetylation of Example 10.Yield: 29 mg (38% overall). LRMS (ESI⁺) m/z 307.1 (M-OAc)⁺, 389.0(M+Na)⁺.

6-Bromo-fucose Diacetonide (23)

Referring to Scheme 7, the alcohol (Compound 2) (150 mg, 0.58 mmol) wasdissolved in DMF (2 mL) followed by addition of PPh₃ (0.61 g, 2.3 mmol,4 eq.). N-bromosuccinimide (0.41 g, 2.3 mmol, 4 eq.) in DMF (1 mL) wasadded over 5 min via syringe. The mixture was heated to 110° C. for 2 h.The reaction was cooled and quenched with MeOH and stirred for 10 min.Ether and sat. NaHCO₃ were added and the layers separated. The aqueouslayer was further washed with ether and the combined organic extractswere washed with water and dried (Na₂SO₄). The solvent was evaporatedand the residue purified by FCC (9:1 hexanes-EtOAc) to afford theproduct as a sticky solid. Yield: 123 mg (66%). LRMS (ESI⁺) m/z 323(M+H)⁺. ¹H-NMR (CDCl₃) δ: 1.34 (s, 3H), 1.36 (s, 3H), 1.45 (s, 3H), 1.55(s, 3H), 3.43 (dd, J=6.8 Hz, 10 Hz, 1H), 3.52 (dd, J=6.8 Hz, 10.4 Hz,1H), 3.98 (dt, J=2.0 Hz, 6.8 Hz, 1H), 4.33 (dd, J=2.4 Hz, 5.2 Hz, 1H),4.38 (dd, J=2.0 Hz, 8.0 Hz, 1H), 4.64 (dd, J=2.4 Hz, 8.0 Hz, 1H), 5.55(d, J=5.2 Hz).

6-Bromo-fucose Tetraacetate (24)

Compound 24 was prepared from Compound 23 following the generalprocedure for acetonide hydrolysis and peracetylation of Example 10.Yield: 129 mg (86% overall). LRMS (ESI⁺) m/z 351.0 (M-OAc)⁺, 432.9(M+Na)⁺.

6-Iodo-fucose Diacetonide (25)

Referring to Scheme 7, the protected sugar (Compound 2) (0.44 g, 1.7mmol), PPh₃ (0.99 g, 3.7 mmol, 2.2 eq.), iodine (0.87 g, 3.4 mmol, 2.0eq.), and imidazole (0.51 g, 7.4 mmol, 4.4 eq.) were dissolved intoluene/EtOAc (4 mL/2 mL). The mixture was heated to 90° C. for 6 hwhile stirring. The mixture was cooled in an ice bath, diluted withCHCl₃ and extracted with sat. NaHCO₃. The organic layer was dried(Na₂SO₄), filtered and concentrated to an oil. The residue was purifiedby FCC eluting with hexanes-EtOAc (95:5 to 90:10 gradient). The productwas isolated as a clear oil. Yield: 0.27 g (43%). LRMS (ESI⁺) m/z 371.1(M+H)⁺. ¹H-NMR (CDCl₃) δ: 1.32 (s, 3H), 1.34 (s, 3H), 1.44 (s, 3H), 1.53(s, 3H), 3.20 (dd, J=7.2 Hz, 9.6 Hz, 1H), 3.31 (dd, J=7.2 Hz, 9.6 Hz,1H), 3.94 (dt, J=1.6 Hz, 7.2 Hz, 1H), 4.29 (dd, J=2.4 Hz, 5.0 Hz, 1H),4.40 (dd, J=2.0 Hz, 8.0 Hz, 1H), 4.60 (dd, J=2.4 Hz, 7.8 Hz, 1H), 5.53(d, J=4.8 Hz).

6-Iodo-fucose Tetraacetate (26)

Compound 26 was prepared from Compound 25 following the generalprocedure for acetonide hydrolysis and peracetylation of Example 10.Yield: 30.5 mg (75% overall). LRMS (ESI⁺) m/z 399.0 (M-OAc)⁺.

Example 17: Synthesis of 6-cyano-fucose Tetraacetate 6-Cyano-fucoseDiacetonide (27)

Compound 27 was prepared following a procedure by Streicher and Wunsch(Carbohydr. Res. 338(22): 2375-85 (2003)). Referring to Scheme 7,iodo-galactose (120 mg, 0.32 mmol) and NaCN (51 mg, 1 M) were heated to110° C. in DMF for 12 h. The mixture was cooled, partitioned withCH₂Cl₂-water and the layers separated. The aqueous layer was furtherwashed with CH₂Cl₂ (2×) and the combined organics washed with brine,dried (Na₂SO₄), filtered and concentrated to a brown oil. FCCpurification (9:1 to 4:1 hexanes-EtOAc gradient) led to the pureproduct. Yield: 10 mg (12%). ¹H-NMR (CDCl₃) δ: 1.33 (s, 3H), 1.35 (s,3H), 1.45 (s, 3H), 1.54 (s, 3H), 2.65 (dd, J=6.8 Hz, 16 Hz, 1H), 2.70(dd, J=6.8 Hz, 16 Hz, 1H), 4.05 (dt, J=2.0 Hz, 7.2 Hz, 1H), 4.24 (dd,J=2.0 Hz, 8.0 Hz, 1H), 4.34 (dd, J=2.8 Hz, 4.8 Hz, 1H), 4.65 (dd, J=2.8Hz, 8.0 Hz, 1H), 5.50 (d, J=5.2 Hz).

6-Cyano-arabinose Tetraacetate (28)

Compound 28 was prepared from Compound 27 following the generalprocedure for acetonide hydrolysis and peracetylation of Example 10.Yield: 13 mg (98% overall). LRMS (ESI⁺) m/z 298.0 (M-OAc)⁺, 380.1(M+Na)⁺.

Example 18: Synthesis of Carboxyfucose Tetraacetate CarboxyarabinoseDiacetonide (29)

Following a procedure for the epimer (Bentama, El Hadrami et al., AminoAcids 24(4):423-6 (2003)), the alcohol (Compound 2) (3.44 g, 13.22 mmol)was diluted in 0.5 M NaOH (80 mL, 40 mmol, 3 eq.). After 15 min, KMnO₄(5.22 g, 33.04, 2.5 eq.), dissolved in 106 mL of water, was added. Thereaction stirred for 18 h and the solid filtered off. The filtrate wasextracted with EtOAc (3×) and organic layers discarded. The aqueouslayer was acidified with 1M HCl to pH 2 and extracted with EtOAc (3×).The combined organic layers were dried (MgSO₄), filtered, andconcentrated to give a white solid that needed no further purification.Yield: 3.1 g (86%). LRMS (ESI⁻) m/z 273.2 (M−H)⁻. ¹H-NMR (CDCl₃) δ: 1.36(s, 6H), 1.46 (s, 3H), 1.54 (s, 3H), 4.40 (dd, J=2.4 Hz, 4.8 Hz, 1H),4.70 (d, J=2.0 Hz, 1H), 4.64 (dd, J=2.4 Hz, 8.0 Hz, 1H), 5.50 (d, J=4.8Hz).

Carboxyarabinose Tetraacetate (30)

Compound 30 was prepared from Compound 29 following the generalprocedure for acetonide hydrolysis and peracetylation of Example 10.

Example 19: Synthesis of Carboxymethylarabinose TetraacetateCarboxymethylarabinose Diacetonide (31)

The acid (Compound 29) (100 mg, 0.365 mmol) was dissolved in MeOH (3.65mL, 0.1 M) and cooled to 0° C. After 15 min, 1 M TMSCHN₂ in ether (1.82mL, 5 eq.) was added dropwise via syringe over 15 min. No startingmaterial was detected after 30 min. The reaction was quenched with 5%HOAc/MeOH and the contents evaporated to dryness. Yield: Quant. LRMS(ESI⁺) m/z 289.1 (M+H)⁺. ¹H-NMR (CDCl₃) δ: 1.34 (s, 6H), 1.46 (s, 3H),1.53 (s, 3H), 3.83 (s, 3H), 4.39 (dd, J=2.4 Hz, 5.2 Hz, 1H), 4.59 (dd,J=2.4 Hz, 7.6 Hz, 1H), 4.67 (dd, J=2.4 Hz, 7.6 Hz, 1H), 5.67 (d, J=5.2Hz).

Carboxymethyl-Arabinose Tetraacetate (32)

Compound 32 was prepared from Compound 31 following the generalprocedure for acetonide hydrolysis and peracetylation of Example 10.Yield: 105 mg (77% overall). LRMS (ESI⁺) m/z. 317.0 (M-OAc)⁺, 398.9(M+Na)⁺.

Example 20: Synthesis of 5-methyl-oxiran-arabinose Tetraacetate((3S,4R,5S,6R)-6-((S)-2-methyloxiran-2-yl)-tetrahydro-2H-pyran-2,3,4,5-tetrayltetraacetate) (36)

1-((3aS,5R,5aS,8aR,8bS)-2,2,7,7-tetramethyltetrahydro-3aH-bis[1,3]dioxolo[4,5-b:4′,5′-d]pyran-5-yl)ethanone(33)

Referring to Scheme 8, to a mixture of the alcohol (Compound 2) (236 mg,0.86 mmol) in DCM (10 mL) was added Dess-Martin periodinane (DMP; 438mg, 1.03 mmol)). After several hours an additional portion DMP (100 mg,0.23 mmol) was added, and the mixture was stirred for an additional 1 h.The mixture was directly aspirated onto a 1 mm radial chromatotron plateand eluted with 25% ethyl acetate in hexanes. The first major materialoff the plate was desired product (Rf=0.6; 25% ethyl acetate inhexanes). Yield: 190 mg (81%): LRMS (ESI⁺) m/z 272; ¹H-NMR (CDCl³) δ:5.64 (d, J=5.1 Hz, 1H), 4.63 (dd, J=7.8, 2.6 Hz, 1H), 4.55 (dd, J=7.8,2.3 Hz), 4.35 (dd, J=5.1, 2.5 Hz), 4.17 (d, J=2.0 Hz, 1H), 2.26 (s, 3H),1.5 (2, 3H), 1.44 (s, 3H), 1.34 (s, 3H), 1.31 (s, 3H).

(3aS,5S,5aR,8aR,8bS)-2,2,7,7-tetramethyl-5-(prop-1-en-2-yl)tetrahydro-3aH-bis[1,3]dioxolo[4,5-b:4′,5′-d]pyran(34)

Referring again to Scheme 8, Compound 34 was prepared from Compound 33(50 mg, 0.18 mmol) in a fashion similar to that used in preparation ofCompound 11, to give 19 mg (39%): ¹H-NMR (CDCl₃) δ: 5.61 (d, J=5.1 Hz,1H), 5.10 (d, J=2.1 Hz, 1H), 4.99 (dd, J=3.1, 1.6 Hz, 1H), 4.34 (m, 2H),4.19 (s, 1H), 1.82 (s, 3H), 1.52 (s, 3H), 1.45 (s, 3H), 1.34 (s, 6H).

(3S,4R,5R,6S)-6-(prop-1-en-2-yl)-tetrahydro-2H-pyran-2,3,4,5-tetrayltetraacetate (35)

Referring again to Scheme 8, Compound 35 was prepared from Compound 34(11 mg, 0.04 mmol) following the general procedure for acetonidehydrolysis and peracetylation of Example 10. Yield 81% (11.7 mg, 0.033mmol)

(3S,4R,5S,6R)-6-((S)-2-methyloxiran-2-yl)-tetrahydro-2H-pyran-2,3,4,5-tetrayltetraacetate (36)

Referring to Scheme 8, to a mixture of the peracetate (Compound 35) (6mg, 0.017 mmol) in DCM (1 mL) was added m-CPBA (12 mg, 0.052 mmol) andthe mixture was stirred at an ambient temperature for 16 hours. Thereaction mixture was aspirated onto 1 mm radial chromatotron plate andeluted with 25% ethyl acetate in hexanes to give 4.6 mg (72%) of theepoxide. LRMS (ESI+) m/z 397 (M+Na)+.

Example 21: Synthesis of propargyl fucose tetraacetate((3S,4R,5R,6S)-6-(prop-2-ynyl)-tetrahydro-2H-pyran-2,3,4,5-tetrayltetraacetate) (37)

At −40° C., trifluoromethanesulfonic anhydride (166 μL, 0.98 mmol, 1.5eq.) was added over 2 min via syringe to a solution of the protectedgalactose (Compound 2) (170 mg, 0.65 mmol) and 2,6-lutidine (96 μL, 0.82mmol, 1.25 eq.) in methylene chloride (3 mL). The starting material wasconsumed in 1 h, and the reaction was quenched with sat. NaHCO₃. Themixture was extracted with ether (3×) and the combined organic layersdried (MgSO₄), filtered, and concentrated. The crude product waspurified by flash chromatography (eluting with 9:1 hexanes-EtOAc) toafford the product as a clear oil. The triflate was immediately used inthe next step.

nBuLi (0.70 mL, 1.74 mmol, 2.6 M, 3.8 eq.) was added dropwise to asolution of trimethylsilylacetylene (0.23 mL, 1.61 mmol, 3.5 eq.) andHMPA (85 μL) in THF (1.5 mL) at −60° C. After 15 min of stirring, thetriflate (180 mg, 0.46 mmol) was added and the contents stirred whilewarming to room temperature. After stirring overnight, the reaction wasquenched with saturated NH₄Cl and the mixture extracted with ether (2×).The combined organic layers were dried and concentrated. By LC/MSpartial TMS cleavage occurred. Purification was performed using flashchromatography (eluting with 95:5 to 9:1 hexanes-EtOAc) and bothproducts were collected and concentrated to a clear oil. Overall yield:61 mg (TMS protected), 68 mg (deprotected), 58% yield. TMS protecteddata: LRMS (ESI+) m/z 341.1 (M+H)+, 363.1 (M+Na)+. 1H-NMR (CDCl3) δ:0.15 (s, 9H), 1.34 (s, 3H), 1.36 (s, 3H), 1.45 (s, 3H), 1.56 (s, 3H),2.52 (dd, J=6.4 Hz, 16.8 Hz, 1H), 2.63 (dd, J=8.4 Hz, 16.4 Hz, 1H), 3.91(dt, J=1.6 Hz, 6.0 Hz, 1H), 4.30 (dd, J=2.4 Hz, 4.8 Hz, 1H), 4.32 (dd,J=2.0 Hz, 8.0 Hz, 1H), 4.62 (dd, J=2.4 Hz, 8.0 Hz, 1H), 5.50 (d, J=5.2Hz, 1H).

The combined alkynes were deprotected using TFA (1 mL) and water (100μL) for 2 h. The mixture was concentrated under high vacuum andperacetylated with acetic anhydride (1 mL), pyridine (1 mL), and DMAP (3mg), for 3 days. The mixture was concentrated and purified by flashchromatography (eluting with 4:1 to 3:2 hexanes-EtOAc). The desiredfractions were pooled and concentrated to give the product as a clearsticky solid. Overall yield: 32 mg (51%). LRMS (ESI+) m/z 297.1(M-OAc)+, 379.0 (M+Na)+.

Example 22: Synthesis of alkynyl fucose tetrapropanoate((3S,4R,5R)-5-((S)-1-(propionyloxy)prop-2-ynyl)-tetrahydrofuran-2,3,4-triyltripropionate mixture) (38)

To a mixture of Compound 6 (25 mg, 0.143 mmol) in pyridine was addedacid chloride (1 mL, propionyl chloride). The reaction mixturesolidified and DCM (2 mL), and DMAP (5 mg) was added and the mixture wasstirred overnight at an ambient temperature. The reaction mixture wastreated with saturated aqueous sodium bicarbonate with stirring for −10min. The reaction mixture was poured into water and extracted with ethylacetate (3×25 mL). The combined extracts were washed with 1N HCl (20mL), saturated aqueous sodium bicarbonate (20 mL) and brine before beingdried over MgSO₄, filtered and concentrated. The resulting residue waspurified via radial chromatography on a 1 mm plate eluting with 25%ethyl acetate in hexanes to give a heterogeneous mixture of α andβ-pyranose and furanose isomers. Yield: 26.8 mg (47%). LRMS (ESI⁺) m/z421 (M+Na+), 325 (M-propionate)⁺.

Example 23: Synthesis of Alkynyl Fucose tetra-n-hexanoates(3S,4R,5R)-5-((S)-1-(hexanoyloxy)prop-2-ynyl)-tetrahydrofuran-2,3,4-triyltrihexanoate and(2S,3S,4R,5R,6S)-6-ethynyl-tetrahydro-2H-pyran-2,3,4,5-tetrayltetrahexanoate mixture (39 and 40, respectively); and(2R,3S,4R,5R,6S)-6-ethynyl-tetrahydro-2H-pyran-2,3,4,5-tetrayltetrahexanoate (41)

Referring to Scheme 9, to a mixture of Compound 6 (25 mg, 0.143 mmol) inpyridine (1 mL) was added DMAP (˜5 mg) and hexanoic anhydride (1 mL).The mixture was stirred overnight at an ambient temperature. Thereaction mixture was treated with saturated aqueous sodium bicarbonatewith stirring for ˜10 min., and the reaction mixture was poured intowater and extracted with ethyl acetate (3×25 mL). The combined extractswere washed with 1N HCl (20 mL), saturated aqueous sodium bicarbonate(20 mL) and brine before being dried over MgSO₄, filtered andconcentrated. The resulting residue was purified via radialchromatography on a 1 mm plate eluting with 25% ethyl acetate hexanes togive to products shown in Scheme 9.(3S,4R,5R)-5-((S)-1-(hexanoyloxy)prop-2-ynyl)-tetrahydrofuran-2,3,4-triyltrihexanoate and(2S,3S,4R,5R,6S)-6-ethynyl-tetrahydro-2H-pyran-2,3,4,5-tetrayltetrahexanoate mixture: LRMS (ESI⁺) m/z 589 (M+Na⁺).(2R,3S,4R,5R,6S)-6-ethynyl-tetrahydro-2H-pyran-2,3,4,5-tetrayltetrahexanoate: ¹H-NMR (CDCl₃) δ 5.70 (d, J=8.4 Hz, 1H), 5.52 (dd,J=3.5, 1.2 Hz, 1H), 5.35 (t, J=8.2 Hz, 1H), 5.1 (dd, J=8.2, 3.4 Hz, 1H),4.60 (dd, J=2.4, 0.6 Hz, 1H), 2.50-1.90 (m, 11H), 1.70-1.50 (m, 9H),1.19-1.10 (m, 20H), 0.95-0.83 (m, 15H); LRMS (ESI₊) m/z 589 (M+Na₊).

Example 24: Synthesis of Alkynyl Fucose tetrakis(trimethylacetate)((2S,3S,4R,5R)-5-((S)-1-(pentanoyloxy)prop-2-ynyl)-tetrahydrofuran-2,3,4-triyltripentanoate (42) and(2R,3S,4R,5R)-5-((S)-1-(pentanoyloxy)prop-2-ynyl)-tetrahydrofuran-2,3,4-triyltripentanoate (43)); alkynyl fucose tris(trimethylacetate)(3S,4R,5R,6S)-6-ethynyl-5-hydroxy-tetrahydro-2H-pyran-2,3,4-triyltripentanoate mixture (44); and alkynyl fucose bis(trimethylacetate(2R,3S,4R,5R,6S)-6-ethynyl-3,5-dihydroxy-tetrahydro-2H-pyran-2,4-diyldipentanoate (45))

To a mixture of Compound 6 (see Example 10: 25 mg, 0.143 mmol) inpyridine (1 mL) was added DMAP (˜5 mg) and trimethyl acetic anhydride (1mL). The mixture was stirred overnight at an ambient temperature. Thereaction mixture was treated with saturated aqueous sodium bicarbonatewith stirring for ˜10 min., and the reaction mixture was poured intowater and extracted with ethyl acetate (3×25 mL). The combined extractswere washed with 1N HCl (20 mL), saturated aqueous sodium bicarbonate(20 mL) and brine before being dried over MgSO₄, filtered andconcentrated. The resulting residue was purified via radialchromatography on a 1 mm plate eluting with 25% ethyl acetate hexanes togive to products shown in Scheme 10.

(2S,3S,4R,5R)-5-((S)-1-(pentanoyloxy)prop-2-ynyl)-tetrahydrofuran-2,3,4-triyltripentanoate: ¹H-NMR (CDCl₃) δ 6.31 (d, J=6.5 Hz, 1H), 5.68 (dd, J=7.0,5.8 Hz, 1H), 5.54 (dd, J=9.2, 2.4 Hz, 1H), 5.38 (dd, J=7.0, 4.7 Hz, 1H),4.29 (dd, J=9.2, 5.6 Hz, 1H), 2.4 (d, J=2.3 Hz, 1H), 1.24 (s, 9H), 1.21(s, 9H), 1.20 (s, 9H), 1.19 (s, 9H); LRMS (ESI₊) m/z 533 (M+Na₊), 409.

(2R,3S,4R,5R)-5-((S)-1-(pentanoyloxy)prop-2-ynyl)-tetrahydrofuran-2,3,4-triyltripentanoate: ¹H-NMR (CDCl₃) δ 6.12 (d, J=0.4 Hz, 1H), 5.59 (dd, J=6.8,2.2 Hz, 1H), 5.35 (dt, J=4.1, 1.5 Hz, 1H), 5.03 (dd, J=1.0, 0.4 Hz, 1H),2.46 (d, J=2.3 Hz, 1H), 1.24 (s, 9H), 1.24 (s, 9H), 1.23 (s, 9H), 1.22(s, 3H); LRMS (ESI⁺) m/z 533 (M+Na⁺), 409.

(3 S,4R,5R,6S)-6-ethynyl-5-hydroxy-tetrahydro-2H-pyran-2,3,4-triyltripentanoate mixture: LRMS (ESI⁺) m/z 449 (M+Na⁺).

(2R,3S,4R,5R,6S)-6-ethynyl-3,5-dihydroxy-tetrahydro-2H-pyran-2,4-diyldipentanoate: ¹H-NMR (CDCl₃) δ 5.54 (d, J=8.2 Hz, 1H), 4.84 (dd, J=10.0,3.2 Hz, 1H), 4.52 (dd, J=2.1, 1.1 Hz, 1H), 4.15 (dd, J=3.2, 1.2 Hz, 1H),4.08 (dd, J=10.0, 8.1 Hz, 1H), 2.58 (d, J=2.2 Hz, 1H), 1.26 (s, 9H),1.25 (s, 9H); LRMS (ESI⁺) m/z 365 (M+Na⁺).

Example 25: Synthesis of(3S,4R,5R,6S)-6-ethynyl-tetrahydro-2H-pyran-2,3,4,5-tetrayltetrakis(2-methylpropanoate) (46)

(Followed procedure 2) To a mixture of the tetra-ol (5 mg, 0.028 mmol)in pyridine (0.2 mL) was added DMAP (˜1 mg) and the anhydride (0.2 mL or200 mg). The mixture was stirred overnight at an ambient temperature andwas treated with saturated aqueous sodium bicarbonated with stirring for10 min. The mixture was poured into water and extracted with ethylacetate (3×25 mL). The combined extracts were washed with 1N HCl (20mL), saturated aqueous sodium bicarbonate (20 mL) and brine before beingdried over MgSO₄, filtered and concentrated. The resulting residue waspurified via radial chromatography on a 1 mm plate eluting with 25%ethyl acetate hexanes to give the product in the α-pyranose form: ¹H-NMR(CDCl₃) δ 6.42 (d, J=3 Hz, 1H), 5.6 (s, 1H), 5.4 (m, 1H), 2.8-2.57 (m,2H), 2.50-2.38 (m, 3H), 1.30-1.07 (m, 13H), 1.06-1.02 (m, 11H). LRMS(ESI⁺) m/z 477.1 (M+Na⁺).

Example 26

Procedure 3:

To a solution of the tetra-ol (5 mg, 0.028 mmol) in DMF (100 uL) wasadded nicotinic acid (70 mg, 0.57 mmol), DMAP (0.5 mg) and EDCI-HCl (55mg, 0.28 mmol). The reaction mixture was stirred at an ambient temperatefor 16 h. The mixture was treated with saturated aqueous sodiumbicarbonate (5 mL) with stirring for 5 min. The resulting mixture wasextracted with ethyl acetate (3×3 mL). The combined extracts were washedwith water and brine, dried over MgSO₄, filtered and concentrated underreduced pressure. The mixture was purified via radial chromotography ona 1 mm plate eluting with 5% methanol in methylene chloride. A singlemajor band was collected and concentrated to give the perester.

The following were prepared utilizing the procedure above:

(3S,4R,5R,6S)-6-ethynyl-tetrahydro-2H-pyran-2,3,4,5-tetrayltetranicotinate (47)

Yield 12.1 mg (72%), LRMS (ESI⁺) m/z 594.85 (M+H).

(3S,4R,5R,6S)-6-ethynyl-tetrahydro-2H-pyran-2,3,4,5-tetrayltetrakis(3-(2-methoxyethoxy)propanoate) (48)

Yield 18.5 mg (95%), LRMS (ESI+) m/z 717 (M+Na)+.

(3S,4R,5R,6R)-6-ethynyl-tetrahydro-2,3,4,5-tetrayl tetraisonicotinate(49)

Yield 13.0 mg (78%), LRMS (ESI+) m/z 594 (M+H)+.

Example 27: Preparation of(3S,4R,5R,6S)-6-(benzyloxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetrayltetraacetate

(3aS,5S,5aR,8aR,8bS)-5-(benzyloxymethyl)-2,2,7,7-tetramethyltetrahydro-3aH-bis[1,3]dioxolo[4,5-b:4′,5′-d]pyran(50)

To a mixture of the alcohol (100 mg, 0.38 mmol) and benzyl bromide (83μL, 0.72 mmol) in THF (2 mL) was added NaH (50 mg of a 60% dispersion inmineral oil) and the reaction mixture was stirred overnight at anambient temperature. To the mixture was added sat. aq. NH₄Cl (10 mL) andthe mixture was extracted with ethyl acetate (3×25 mL). The combinedextracts were washed with water and brine and were dried over MgSO₄.Filteration and concentration gave a residue that was purified viaradial chromatography on a 1 mm plate, eluting with 10% ethyl acetate inhexanes to give 63 mg (47%): ¹H-NMR (CDCl₃) δ 7.37-7.22 (m, 5H), 5.55(d, J=4.9 Hz, 1H), 4.64-4.53 (m, 3H), 4.32 (dd, J=5.1, 2.3 Hz, 1H), 4.27(dd, J=7.8, 2.4 Hz, 1H), 4.01 (dt, J=6.4, 1.9 Hz, 1H), 3.72-3.61 (m,2H), 1.54 (s, 3H), 1.44 (s, 3H), 1.33 (s, 6H).

(3S,4R,5R,6S)-6-(benzyloxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetraylTetraacetate (51)

To a round-bottom flask charged with the benzyl ether (63 mg, 0.18 mmol)and cooled to 0° C. was added ice-cold TFA/H2O (9:1, 5 mL). The mixturewas stirred for 1 h and was concentrated under reduced pressure. Theresidue was then treated with pyridine (3 mL), DMAP (5 mg) and aceticanhydride (3 mL). The mixture was stirred 16 h at an ambient temperatureand was concentrated under reduced pressure. The residue was purifiedvia radial chromatography on a 2 mm plate eluting with 25% ethyl acetatein hexanes to give the a mixture of pyranose and furanose benzy etherperacetates, 97 mg (0.22 mmol, 122%): LRMS (ESI+) m/z 378.9 (M-OAc)+.

Example 28: Preparation of(2R,3R,4S)-2-((S)-1-acetoxyprop-2-ynyl)-5-methoxy-tetrahydrofuran-3,4-diyldiacetate (52)

(2R,3R,4S)-2-((S)-1-acetoxyprop-2-ynyl)-5-methoxy-tetrahydrofuran-3,4-diylDiacetate (52)

A round-bottom flask was charged with CH₃OH (2 mL) and propionylchloride (20 μL) was added. After 5 min, the tetra-ol (˜5 mg, 0.028mmol) was added and the mixture was stirred overnight at an ambienttemperature. The mixture was concentrated under reduced pressure, theresidue was treated with pyridine (1 mL), DMAP (0.5 mg) and aceticanhydride (1 mL), stirred for ˜2 h and concentrated under reducedpressure. The resulting residue was purified by radial chromatography togive a mixture of the two furanose triacetates as an inseparablemixture, 6.1 mg (69%): LRMS (ESI+) m/z 336.95 (M+Na)+.

Example 29:(3S,4R,5S,6R)-6-(difluoromethyl)tetrahydro-2H-pyran-2,3,4,5-tetrayltetraacetate

(3aS,5R,5aS,8aR,8bS)-5-(difluoromethyl)-2,2,7,7-tetramethyltetrahydro-3aH-bis[1,3]dioxolo[4,5-b:4′,5′-d]pyran(53)

A mixture of the aldehyde (70 mg, 0.23 mmol) and absolute ethanol (3.1μL, 54 μmol, 0.2 eq.) in methylene chloride (115 μL, 2 M) was treatedwith bis (2-methoxyethyl)aminosulfur trifluoride (Deoxo-fluor, 85 μL,0.46 mmol, 1.7 eq.) in a sealed Eppendorf tube. The contents stood at37° C. for 72 h. The reaction was cooled and then purified by flashchromatography (eluting with 9:1 to 4:1 hexanes-EtOAc). Thedifluoro-diacetonide intermediate was isolated as a clear oil. Yield: 35mg, 46% yield. ¹H-NMR (CDCl₃) δ: 1.34 (s, 3H), 1.35 (s, 3H), 1.46 (s,3H), 1.54 (s, 3H), 3.85-3.92 (m, 1H), 4.33-4.38 (m, 2H), 4.62-4.67 (m,1H), 5.56 (dd, J=2 Hz, 4.8 Hz, 1H), 5.84 (dt, J=6.8 Hz, 54 Hz, 1H).

(3S,4R,5S,6R)-6-(difluoromethyl)tetrahydro-2H-pyran-2,3,4,5-tetraylTetraacetate (54)

The aforementioned compound (30 mg, 0.11 mmol) was treated withtrifluoroacetic acid (1 mL) and water (100 uL) for 2 h. The mixture wasconcentrated under high vacuum and peracetylated with acetic anhydride(1 mL), pyridine (1 mL), and DMAP (5 mg), for 1 d. The mixture wasconcentrated and purified by flash chromatography (eluting with 4:1 to1:1 hexanes-EtOAc). The desired fractions were pooled and concentratedto give the product as a clear sticky solid. Overall yield: 24 mg (62%).LRMS (ESI⁺) m/z 309 (M-OAc)⁺, 391 (M+Na)⁺.

Example 30: Preparation of 2-Fluoro-2-Deoxyfucose Peracetate (58)

Compounds 56, 57 and 58 were prepared according to the followingreferences:

-   1. Oberthur, M.; Leimkuhler, C.; Kuguer, R. G.; Lu, W.; Walsh, C.    T.; Kahne, D. J. Am. Chem. Soc. 2005, 127, 10747-10752-   2. a) Murray, B. W.; Wittmann, V.; Burkhart, M. D.; Hung, S-C.;    Wong, C-H. Biochemistry, 1997, 36, 823-831. b) Korytnky, W.;    Valentekovic-Horvath, S.; Petrie, C. R. Tetrahedron, 1982, 38(16),    2547-2550.

Example 31: Preparation of(3S,4R,5R,6S)-6-(propa-1,2-dienyl)-tetrahydro-2H-pyran-2,3,4,5-tetrayltetraacetate (60)

Allenyl Diacetonide (59)

To a suspension of alkyne (compound 5, 25 mg, 0.1 mmol),paraformaldehyde (7 mg, 0.215 mmol), CuBr (5 mg, 0.035 mmol) and dioxane(0.5 mL) in a pressure tube was added DIPEA (28 μL, 0.223 mmol). Thepressure tube was sealed and the brown mixture was heated at reflux for16 h then cooled to rt and filtered. The solid was washed with Et₂O, andthe combined filtrates were concentrated under reduced pressure.Purification by flash chromatography (25% ethyl acetate in hexanes)afforded the desired allene compound 59, 2.3 mg (9%): ¹H NMR (CDCl₃; 400mHz) δ: 5.56 (d, J=4.0 Hz, 1H), 5.36 (q, J=8.0 Hz, 1H), 4.84 (m, 2H),4.62 (dd, J=7.8 Hz, 3.4 Hz, 1H), 4.37 (dd, J=8.2 Hz, 1.7 Hz, 1H), 4.32(d, 1H, J=2.3 Hz, 1H), 1.54 (s, 3H), 1.49 (s, 3H), 1.36 (s, 3H), 1.34(s, 3H).

To the acetonide (compound 59, 2.3 mg, 8.5 mmol) in a round-bottom flaskand cooled in an ice-bath was added ice-cold 10% H₂O/TFA (4 mL) and themixture was stirred for 1 h. After concentration under reduced pressure,the resulting residue was treated with pyridine (2 mL), DMAP (0.5 mg)and acetic anhydride (2 mL). The reaction mixture was stirred overnightand the mixture was concentrated under reduced pressure and purified byradial chromatography on a 1 mm plate eluting with 25% ethyl acetate inhexanes. A single band was collected and concentrated to give 4.1 mgcompound 60 as a mixture of anomeric acetates: LRMS (ESI⁺) m/z 378.98(M+Na⁺)

Example 32 Preparation of 2-Fluoro-2-Deoxyfucose Peracetate

Preparation of 61

To a solution of compound 56 (500 mg, 2.3 mmol) in DMF/H₂O (30 mL of a1:1 mixture) was added Selectfluor® (1.24 g, 3.5 mmol) and the mixturewas stirred at an ambient temperature for 12 h. The mixture was dilutedwith ethyl acetate (100 mL) and washed with water (3×100 mL). Theorganic layer was dried over Na₂SO₄, filtered and concentrated underreduced pressure: LRMS (ESI⁺) m/z 273.04 (M+Na⁺). See publishedprocedure: Burkart, M. D.; Zhang, Z.; Hung, S-C.; Wong, C-H. J. Am.Chem. Soc. 1997, 119, 11743-11746.

(3S,4R,5R,6S)-3-fluoro-6-methyl-tetrahydro-2H-pyran-2,4,5-triylTriacetate (58)

To a mixture of compound 61 in pyridine (10 mL) was added aceticanhydride (10 mL) followed by DMAP (10 mg) and the mixture was stirredfor 2 h at an ambient temperature. The mixture was concentrated underreduced pressure, dissolved in DCM (5 mL) and aspirated onto a 2 mmradial chromatotron plate; eluting with 25% ethyl acetate in hexanes. Asingle band was collected and concentrated to give 420 mg of compound 58(1.44 mmol, 63%) as an inseparable mixture of anomers (α/β=36.64): ¹HNMR (CDCl₃; 400 mHz) δ (α-anomer): 6.43 (d, J=4.11 Hz, 1H), 5.41 (dt,J=10.8, 3.72 Hz, 1H), 5.37 (m, 1H), 4.88 (ddd, J=49.5, 10.2, 3.9 Hz,1H), 4.25 (q, 1H, J=6.7 Hz, 1H), 2.9 (s, 3H), 2.07 (s, 3H), 1.15 (d,J=6.5 Hz, 3H); β-anomer: 5.77 (dd, J=8.02, 4.2 Hz, 1H), 5.3 (m, 1H),5.17 (dq, J=9.8, 3.5 Hz, 1H), 4.64 (ddd, J=51.8, 9.8, 8.0 Hz, 1H), 3.98(dq, J=6.4 Hz, 1.0 Hz, 1H), 2.18 (s, 3H), 2.07 (s, 3H), 1.22 (d, J=6.2Hz, 3H); LRMS (ESI⁺) m/z 315.02 (M+Na⁺).

Example 33: Preparation ofL-2-deoxy-2-chlorofucopyranose-1,3,4-triacetate, 62

L-2-deoxy-2-chlorofucopyranose-1,3,4-triacetate

To a mixture of the compound 56 (100 mg, 0.47 mmol) in DMF/H₂O (2 mL ofa 1:1 mixture) was added N-chlorosuccinimide (91 mg, 0.7 mmol) and themixture was stirred for 16 h at an ambient temperature. The reactionmixture was poured into ethyl acetate (100 mL) and washed with water andbrine, dried over MgSO₄, filtered and concentrated under reducedpressure. The mixture was taken up in pyridine (2 mL). DMAP (2 mg) wasadded and acetic anhydride (2 mL) was added. The mixture was stirred for16 h at an ambient temperature before being concentrated and purifiedvia radial chromatography on a 1 mm plate eluting with 25% ethyl acetatein hexanes to give 98 mg (0.79 mmol, 79%) of the 2-deoxy-2-chlorofucosetriacetate 62 as a mixture of anomers (α/β=0.73/1.0) as determined by 1HNMR: LRMS (ESI⁺) m/z 330.98 (M+Na⁺).

Example 34: Preparation of(2S,4R,5R,6S)-3,3-difluoro-6-methyl-tetrahydro-2H-pyran-2,4,5-triyltriacetate (65) and(2R,4R,5R,6S)-3,3-difluoro-6-methyl-tetrahydro-2H-pyran-2,4,5-triyltriacetate (66)

1-α-bromofucopyranose-3,4-diacetate (63)

To the 2-fluorofucose triacetate (compound 58, 300 mg, 1.027 mmol) inCH₂Cl₂ (1 mL) was added 33% HBr in HOAc (0.25 mL). The mixture wasstirred for 2 h and was poured into ice-water (100 mL) and extracted(3×50 mL) with DCM. The combined extracts were washed with water anddried with MgSO₄. Filtration and concentration gave 0.313 g (1.0 mmol,98%) of the L-α-1-bromofucopyranoside-3,4-diacetate (63). The materialwas carried forward without purification: ¹H NMR (CDCl₃; 400 mHz) δ 6.60(d, J=4.3 Hz, 1H), 5.48 (dt, J=10.0, 3.5 Hz, 1H), 5.39 (m, 1H), 4.74(ddd, J=50.5, 10.2, 4.3 Hz, 1H), 4.44 (dq, J=5.9, 1.3 Hz), 2.17 (s, 3H),2.06 (s, 3H), 1.22 (d, J=6.4 Hz, 3H).

2-fluorofucal-3,4-diacetate, 64

To a mixture of the bromide (63, 312 mg, 1 mmol) in acetonitrile (10 mL)was added Et₃N (500 μL, 3 mmol) and the reaction mixture was heated toreflux. The reaction was monitored by TLC. After 2 h, the reactionmixture was poured into ethyl acetate (100 mL) and washed with 1N HCl,water and brine and dried over MgSO₄. Filtration and concentration gavea residue that was purified by radial chromatography on a 2 mm plateeluting with 25% ethyl acetate hexanes to give 73 mg (32%): ¹H NMR(CDCl₃; 400 mHz) δ: 6.74 (dd, J=4.9, 1.2 Hz, 1H), 5.97 (dd, J=3.9, 1.2Hz, 1H), 5.3 (dt, J=5.3, 1.4 Hz, 1H), 4.15 (q, J=6.7 Hz, 1H), 2.18 (s,3H), 2.07 (s, 3H), 1.56 (s, 3H), 1.22 (d, J=6.5 Hz, 3H).

2-deoxy-2,2-difluorofucopyranose-1,3,4-triacetate (65 and 66

To a mixture of the fluorofucal (64, 50 mg, 0.216 mmol) in DMF/H₂O (1mL, 1:1 mixture) was added Selectfluor® and the reaction mixture wasstirred overnight at an ambient temperature. The reaction mixture waspoured into EtOAc (100 mL) and washed with water (3×50 mL) and brine,dried over NaSO₄, decanted and concentrated. The resulting residue wasacetylated with a mixture of pyridine (1 mL), DMAP (2 mg) and aceticanhydride (1 mL). The mixture was stirred for several hours andconcentrated under reduced pressure and purified on a 1 mm radialchromatotron plate eluting with 10% ethyl acetate in hexanes to give amixture of anomeric 2-deoxy-2, 2-difluorofucose-1,3,4-diacetates.α-anomer (65): ¹H NMR (CDCl₃; 400 mHz) δ 6.21 (d, J=7.2, 1H), 5.43 (m,1H), 5.33 (m, 1H), 4.33 (dq, J=6.5, 0.9 Hz), 2.19 (s, 6H), 2.12 (s, 3H),1.22 (d, J=6.6 Hz, 3H); LRMS (ESI⁺) m/z 332.90 (M+Na⁺). β-anomer (66):¹H NMR (CDCl₃; 400 mHz) δ 5.78 (d, J=15.5 Hz, 1H), 5.3 (m, 1H), 5.24 (m,1H), 4.06 (dq, J=6.5, 1.4 Hz), 2.23 (s, 3H), 2.19 (s, 3H), 2.12 (s, 3H),1.29 (d, J=6.5 Hz, 1H); LRMS (ESI⁺) m/z 332.99 (M+Na⁺).

Example 35: Preparation of(2S,4S,5R,6S)-6-methyl-tetrahydro-2H-pyran-2,4,5-triyl triacetate

(2S,4S,5R,6S)-6-methyl-tetrahydro-2H-pyran-2,4,5-triyl Triacetate

To a flame-dried flask maintained under a nitrogen atmosphere was addedfucal-3,4-diacetate (110 mg, 0.51 mmol) dissolved in 2.6 mL of anhydrousacetonitrile. Ceric(IV)ammonium nitrate (727 mg, 1.33 mmol) and glacialacetic acid (290 μL, 5.1 mmol) were added and the reaction mixture wasthen cooled to −15° C. Sodium cyanide (33 mg, 0.66 mmol) was then addedand the reaction was stirred at 15° C. under nitrogen for 8 h. Thereaction was quenched with 0.1 M sodium thiosulfate (50 mL). The aqueouslayer was extracted with dichloromethane (3×50 mL) and the combinedorganic layer was washed with brine, dried over sodium sulfate, filteredand concentrated. The residue was purified by flash chromatography onsilica gel eluted with a hexane: ethyl acetate solvent mixture (90:10 to75:25) to provide the title compound (8 mg, 5%). TLC (SiO₂, 3:1hexanes/ethyl acetate): R_(f)=0.20. ¹H NMR (CDCl₃; 400 MHz) δ: 6.29 (m,1H), 5.29 (ddd, J=12.4, 4.8, 2.8 Hz, 1H), 5.22 (m, 1H), 4.17 (q, J=6.8Hz, 1H), 2.19 (m, 1H), 2.17 (s, 3H), 2.11 (s, 3H), 2.01 (s, 3H), 1.88(ddt, J=13.6, 4.8, 1.2, 1 H), 1.15 (d, J=6.8 Hz).

Example 35: Activity of Fucose Analogs

The effects of fucose analogs on antibody core fucosylation were testedat concentrations of 50 μM and 1 mM as follows: A CHO DG44 cell lineproducing a humanized IgG1 anti-CD70 monoclonal antibody, h1F6 (seeInternational Patent Publication WO 06/113909) was cultured at 7.5×10⁵cells per mL in 2 mLs of CHO culture media at 37°, 5% CO₂ and shaking at100 RPM in a 6 well tissue culture plate. Media was supplemented withinsulin like growth factor (IGF), penicillin, streptomycin and either 1mM or 50 μM of the fucose analog (prepared as described supra). On day 5post inoculation, the culture was centrifuged at 13000 RPM for 5 minutesto pellet cells; antibodies were then purified from supernatant.

Antibody purification was performed by applying the conditioned media toprotein A resin pre-equilibrated with 1× phosphate buffered saline(PBS), pH 7.4. After washing resin with 20 resin bed volumes of 1×PBS,antibodies were eluted with 5 resin bed volumes of Immunopure IgGelution buffer (Pierce Biotechnology, Rockford, Ill.). A 10% volume of1M tris pH 8.0 was added to neutralize the eluted fraction. The amountof non-core fucosylated antibody produced was determined as described inExample 7. The results are shown in the following tables.

TABLE 1 Name Inhibition Inhibition (Chemical name) R⁵ R¹-R⁴ at 50 μM at1 mM Alkynyl fucose —C≡CH —OH >80% ND (5-ethynylarabinose) Alkynylfucose peracetate —C≡CH —OAc >80% >80% Alkynyl fucose tetraacetate(5-ethynylarabinose tetraacetate) 5-propynyl fucose tetraacetate —C≡CCH₃—OAc  50% >80% (5-propynylarabinose tetraacetate) propargyl fucosetetraacetate —CH₂C≡CH —OAc ~10% ~10-20%  ((3S,4R,5R,6S)-6-(prop-2-ynyl)- tetrahydro-2H-pyran-2,3,4,5- tetrayltetraacetate) Peracetyl galactose —OAc —OAc  ~0%  ~0% (galactosepentaacetate) 5-vinyl fucose tetraacetate —CHCH₂ —OAc  ~0%  ~4%(5-ethylenylarabinose tetraacetate) 6-cyano fucose tetraacetate —CH₂CN—OAc  30% >80% (6-cyanofucose tetraacetate) 5-cyano fucose tetraacetate—CN —OAc  20% ND (pyranose form) (5-cyanoarabinopyranose tetraacetate)5-cyano fucose tetraacetate —CN —OAc 5-10%  ND (furanose form)(5-cyanoarabinofuranose tetraacetate) 5-methylester fucose —C(O)OCH₃—OAc  30% >80% tetraacetate (5-carboxymethyl arabinose tetraacetate)5-(CH(OAc)CH₃) peracetyl —CH(OAc)CH₃ —OAc  ~0%  40% fucose(6-methylgalactose pentaacetate) 5-methyloxiran-arabinose tetraacetate((3S,4R,5S,6R)-6-((S)-2- methyloxiran-2-yl)-tetrahydro-2H-pyran-2,3,4,5-tetrayl tetraacetate)

—OAc  ~0% ~35-40%   6-iodo-fucose tetraacetate —CH₂I —OAc  3%  30%(6-iodofucose tetraacetate) 6-chloro-fucose tetraacetate —CH₂Cl —OAc 20% 20-30%  (6-chlorofucose tetraacetate 6-bromo-fucose tetraacetate—CH₂Br —OAc  50%  80% (6-bromofucose tetraacetate) Alkynyl fucosetetrapropanonate —C≡CH —OC(O)CH₂—CH₃ >80% >80% (5-ethynylarabinosetetrapropropanoate) Alkynyl fucose tetra-n- —C≡CH—OC(O)(CH₂)₄—CH₃ >80% >80% hexanoate (5-ethynylarabinose tetrahexanoate)Alkynyl fucose —C≡CH —OC(O)C(CH₃)₃  20%  60% tetrakis(trimethylacetate)(5-ethynylarabinose tetra(trimethylacetate)) Alkynyl fucose —C≡CH—OC(O)C(CH₃)₃  5%  10% tetrakis(trimethylacetate) (5-ethynylarabinosetetra(trimethylacetate)) Alkynyl fucose 1,2,3- —C≡CH —OC(O)C(CH₃)₃  ~0%ND (trimethylacetate) and —OH (5-ethynylarabinose 1,2,3-(trimethylacetate)) Alkynyl fucose —C≡CH —OC(O)C(CH₃)₃ >80% NDdi(trimethylacetate) and —OH (5-ethynylarabinose 1,3-(trimethylacetate)) Alkynyl fucose pernicotinate —C≡CH—C(O)-3-pyridyl >80% >80% Alkynyl fucose perisonicotinate —C≡CH—C(O)-4-pyridyl >80% >80% Alkynyl fucose per-PEG ester —C≡CH—C(O)—(CH₂CH₂O)₂—OCH₃ >80% >80% 1-methyl-2,3,4-triacetyl alkynyl —C≡CHR¹ = OCH₃  68% >80% fucose R², R³, R⁴ = OAc Alkynyl fucoseperisobutanoate —C≡CH —OC(O)C(CH₃)₂ >80% >80% “ND” means non-corefucosylated antibody was not detected due to poor antibody production orinhibition of cell growth in the presence of the fucose analog.

TABLE 2 Name Inhibition Inhibition (Chemical name) R⁵ R¹ R²/R^(2a)R³/R^(3a) at 50 μM at 1 mM 2-deoxy-2-fluorofucose —CH₃ —OH —F/—H—OAc/—H >80% >80% diacetate (R⁴ = OAc) 2-deoxy-2-chlorofucose —CH₃ —OAc—Cl/—H —OAc/—H  17% >80% triacetate (R⁴ = OAc) Allene —CH═C═CH₂ —OAc—OAc/—H —OAc/—H  23%  34% (R⁴ = OAc) 2-deoxy-2-fluorofucose —CH₃ —OH—F/—H —OH/—H >80% >80% (R⁴ = OH) 2-deoxy-2-fluorofucose —CH₃ —OAc —F/—H—OAc/—H >80% >80% peracetate (R⁴ = OAc) 1,2-difluoro-1,2-didexoy —CH₃ —F—F/—H —OAc/—H >80% >80% fucose peracetate (R⁴ = OAc) 6,6-difluorofucose—CHF₂ —OAc —OAc/—H —OAc/—H >80% >80% tetraacetate (R⁴ = OAc)2-deoxy-2,2- —CH₃ —OAc —F/—F —OAc/—H 0  64% difluorofucopyranosetriacetate (alpha) (R⁴ = OAc) 2-deoxy-2,2- —CH₃ —OAc —F/—F —OAc/—H 0 75% difluorofucopyranose triacetate (beta) (R⁴ = OAc)6-methyl-tetrahydro-2H- —CH₃ —OAc —H/—H —OAc/—H 0  36% pyran-2,4,5-triyltriacetate (R⁴ = OAc) 5-Benzyloxy fucose —CH₂OCH₂Ph —OAc —OAc/—H —OAc/—H0  75% peracetate (R⁴ = OAc) “ND” means non-core fucosylated antibodywas not detected due to poor antibody production or inhibition of cellgrowth in the presence of the fucose analog.

Certain other fucose analogs were tested for their ability to beincorporated into antibodies. These fucose analogs were tested atconcentrations of 50 μM and 1 mM using the methodology as describedabove and in Example 7. The results are shown in the following table.

TABLE 3 Name % Incor- (Chemical name) R⁵ R¹-R⁴ poration Propargyl fucoseor (3S,4R,5R)-6-(prop-2- ynyl)tetrahydro-2H-pyran- 2,3,4,5-tetrayltetraacetate

—OAc 80% (1 mM) 5-(Z)-propenyl fucose peracetate

—OAc ~30% Isopropenyl peracetyl fucose or (3S,4R,5R,6S)-6-(prop-1-en-2-yl)-tetrahydro-2H-pyran-2,3,4,5- tetrayl tetraacetate

—OAc >80% (1 mM and 50 uM) 5-ethyl fucose —CH₃CH₂ —OH >80% (1 or mM and(3S,4R,5S,6S)-6-ethyl- 50 uM) tetrahydro-2H-pyran-2,3,4,5- tetraol5-ethyl fucose peracetate —CH₃CH₂ —OAc >90% (1 or mM and(3S,4R,5S,6S)-6-ethyl- 50 uM) tetrahydro-2H-pyran-2,3,4,5- tetrayltetraacetate 5-cyclopropyl fucose or (3S,4R,5S,6S)-6-cyclopropyltetrahydro-2H-pyran- 2,3,4,5-tetraol

—OH ~80% 5-cyclopropyl fucose peracetate or (3S,4R,5R,6S)-6-cyclopropyltetrahydro-2H-pyran- 2,3,4,5-tetrayl tetraacetate

—OAc ~80% 5-propyloxyarabinose tetraacetate or (3S,4R,5S,6R)-6-((S)-2-methyloxiran-2-yl)tetrahydro- 2H-pyran-2,3,4,5-tetrayl tetraacetate

—OAc ~60% Fluoromethylene fucose —CH₂F —OAc >90% (1 or mM and(3S,4R,5S)-6- 50 uM) (fluoromethyl)tetrahydro-2H- pyran-2,3,4,5-tetrayltetraacetate 5-chloromethylene peracetyl —CH₂Cl —OAc ~80% fucose or(3S,4R,5S)-6- (chloromethyl)tetrahydro-2H- pyran-2,3,4,5-tetrayltetraacetate 5-bromomethylene peracetyl —CH₂Br —OAc ~50% fucose (50 uM;or 20% at 1 (3S,4R,5S)-6- mM) (bromomethyl)tetrahydro-2H-pyran-2,3,4,5-tetrayl tetraacetate 5-iomethylene-peracetyl fucose —CH₂I—OAc ~30% or (3S,4R,5S)-6- (iodomethyl)tetrahydro-2H-pyran-2,3,4,5-tetrayl tetraacetate Azido peracetyl fucose —CH₂N₃ —OAc 60% or (3S,4R,5R)-6- (azidomethyl)tetrahydro-2H- pyran-2,3,4,5-tetrayltetraacetate 5-(2-azidoethyl) arabinose —CH₂CH₂N₃ —OAc  20% tetraacetateor (3S,4R,5R,6S)-6-(2- azidoethyl)tetrahydro-2H-pyran- 2,3,4,5-tetrayltetraacetate —CH═C═CH₂ —OAc ~30% Isopropyl peracetyl fucose Isopropyl—OAc Not or detected (3S,4R,5R,6S)-6- isopropyltetrahydro-2H-pyran-2,3,4,5-tetrayl tetraacetate

Example 36: Titration Method to Determine Effective Levels of FucoseAnalogs

A CHO DG44 cell line producing a humanized IgG1 anti-CD70 monoclonalantibody, h1F6 (see International Patent Publication WO 06/113909) wascultured at 3.0×10⁵ cells per mL in 30 mLs of CHO culture media at 37°,5% CO₂ and shaking at 100 RPM in a 125 mL shake flask. Media wassupplemented with insulin like growth factor (IGF), penicillin,streptomycin and either 100 μM, 50 μM, 5 μM, 500 nM, or 50 nM alkynylfucose peracetate. Cultures were fed on day 3 with 2% volume of a feedmedia containing 5 mM, 2.5 mM, 250 μM, 25 μM, and 2.5 μM alkynyl fucoseperacetate for the respective cultures. On day four, the culture wassplit 1:4 into fresh culture media. Cultures were fed with a 6% volumeof production feed media containing 1.66 mM, 833 μM, 83 μM, 8.3 μM and833 nM alkynyl fucose peracetate, respectively, on days 5, 7, 9 and 10.Supplementation of the feed media is optional. Conditioned media wascollected on day 13 by passing culture through a 0.2 μm filter.

Antibody purification was performed by applying the conditioned media toa protein A column pre-equilibrated with 1× phosphate buffered saline(PBS), pH 7.4. After washing the column with 20 column volumes of 1×PBS,antibodies were eluted with 5 column volumes of Immunopure IgG elutionbuffer (Pierce Biotechnology, Rockford, Ill.). A 10% volume of 1M trispH 8.0 was added to eluted fraction. The sample was dialyzed overnightinto 1×PBS. The carbohydrate composition was determined using capillaryelectrophoresis.

Referring to FIG. 6, the results of a titration of alkynyl fucoseperacetate (“Alk Fuc peracetate”) on a culture of host cells expressingh1F6 antibody and the effect on production of Ab with core fucosylation(G0). As the amount of G0 antibody produced decreased, the amount ofnon-core-fucosylated antibody increased.

Example 37: Non-Core Fucosylated Antibody Production in DifferentCulture Media

To determine the effect of different culture media on non-corefucosylated antibody production, a CHO DG44 cell line producing ahumanized IgG1 anti-CD70 monoclonal antibody, h1F6 (see InternationalPatent Publication WO 06/113909), was cultured in various media. Thecells (7.5×10⁵ cells per mL in 2 mLs) were cultured in PowerCHO (LonzaGroup Ltd., Basil, Switzerland) or OptiCHO (Invitrogen, Carlsbad,Calif.) media CHO culture media at 37°, 5% CO₂ and shaking at 100 RPM ina 6 well tissue culture plate. Media was supplemented with insulin likegrowth factor (IGF), penicillin, streptomycin and 50 μM alkynyl fucoseperacetate. On day 5 post-inoculation, the culture was centrifuged at13000 RPM for 5 minutes to pellet cells; antibodies were then purifiedfrom supernatant.

Antibody purification was performed by applying the conditioned media toprotein A resin pre-equilibrated with 1× phosphate buffered saline(PBS), pH 7.4. After washing resin with 20 resin bed volumes of 1×PBS,antibodies were eluted with 5 resin bed volumes of Immunopure IgGelution buffer (Pierce Biotechnology, Rockford, Ill.). A 10% volume of1M tris pH 8.0 was added to neutralize the eluted fraction. Productionof non-core fucosylated antibody was determined as described in Example7. The proportion of non-core fucosylated to core fucosylated antibodyproduced from each media was similar.

The present invention is not limited in scope by the specificembodiments described herein. Various modifications of the invention inaddition to those described herein will become apparent to those skilledin the art from the foregoing description and accompanying figures. Suchmodifications are intended to fall within the scope of the appendedclaims. Unless otherwise apparent from the context any step, element,embodiment, feature or aspect of the invention can be used incombination with any other. All patent filings, and scientificpublications, accession numbers and the like referred to in thisapplication are hereby incorporated by reference in their entirety forall purposes to the same extent as if so individually denoted.

What is claimed is:
 1. A fucose analog having one of the followingformulae (I) or (II):

or a biologically acceptable salt or solvate thereof, wherein each offormula (I) or (II) can be the alpha or beta anomer or the correspondingaldose form; each of R¹-R⁴ is independently selected from the groupconsisting of —OH, —OC(O)H, —OC(O)C₁-C₁₀ alkyl, —OC(O)aryl,—OC(O)heterocycle, —OC(O)C₁-C₁₀ alkylene aryl, —OC(O)C₁-C₁₀ alkyleneheterocycle, —OCH₂OC(O) alkyl, —OCH₂OC(O)O alkyl, —OCH₂OC(O) aryl,—OCH₂OC(O)O aryl, —OC(O)CH₂O(CH₂CH₂O)_(n)CH₃, and—OC(O)CH₂CH₂O(CH₂CH₂O)_(n)CH₃, wherein each n is an integerindependently selected from 0-5; and R⁵ is selected from the groupconsisting of —C≡CH, —C≡CCH₃, —CH₂C≡CH, —C(O)OCH₃, —CH(OAc)CH₃, —CN,—CH₂CN, —CH₂Br, —CH₂Cl, —CH₂I, and -methoxiran; provided that R⁵ is not—C≡CH when any of R¹-R⁴ is —OH or —OAc, and the fucose analog is otherthan a compound selected from the group consisting of:

the alpha or beta anomer thereof, and the corresponding aldose formthereof.
 2. The fucose analog of claim 1, wherein each of R¹-R⁴ isindependently selected from the group consisting of —OH and —OC(O)C₁-C₁₀alkyl.
 3. The fucose analog of claim 1, wherein each of R¹-R⁴ isindependently selected from the group consisting of —OH and —OAc.
 4. Thefucose analog of claim 1, wherein R⁵ is selected from the groupconsisting of —C≡CH and —C≡CCH₃.
 5. The fucose analog of claim 1,wherein R⁵ is —C(O)OCH₃.
 6. The fucose analog of claim 1, wherein R⁵ isselected from the group consisting of —CN and —CH₂CN.
 7. The fucoseanalog of claim 1, wherein R⁵ is —CH₂CN.
 8. The fucose analog of claim1, wherein R⁵ is —CH₂Br, —CH₂Cl, or —CH₂I.
 9. The fucose analog of claim1, wherein R⁵ is —CH₂Br or —CH₂I.
 10. The fucose analog of claim 1,wherein R⁵ is —CH₂Br.
 11. The fucose analog of claim 1, wherein R⁵ ismethoxiran.
 12. The fucose analog of claim 1, wherein R⁵ is —CH(OAc)CH₃.